nach oben

International Journal of Colorectal Disease

Erschienen in:

Open Access 01.03.2013 | Original Article

Single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer xenograft—no benefit of combination therapy

verfasst von: Thomas C. Wehler, Swaantje Hamdi, Annett Maderer, Claudine Graf, Ines Gockel, Irene Schmidtmann, Michael Hainz, Martin R. Berger, Matthias Theobald, Peter R. Galle, Markus Moehler, Carl C. Schimanski

Erschienen in: International Journal of Colorectal Disease | Ausgabe 3/2013

Abstract

Background

We initiated this preclinical study in order to analyze the impact of sorafenib single treatment versus combination treatment in human colorectal cancer.

Methods

The effect of increasing sorafenib doses on proliferation, apoptosis, migration, and activation of signal cascades was analyzed in vitro. The effect of sorafenib single treatment versus 5-fluorouracil (5-FU) single treatment and combination therapy on in vivo proliferation and target cytokine receptor/ligand expression was analyzed in a human colon cancer xenograft mouse model using HT29 tumor cells.

Results

In vitro, SW480 and HT29 cell lines were sensitive to sorafenib, as compared to Caco2 and SW620 cell lines, independent of the mutation status of K-ras, Raf, PTEN, or PI3K. The effect on migration was marginal, but distinct differences in caspases activation were seen. Combination strategies were beneficial in some settings (sorafenib + 5-FU; irinotecan) and disadvantageous in others (sorafenib + oxaliplatin), depending on the chemotherapeutic drug and cell line chosen. Sensitive cell lines revealed a downregulation of AKT and had a weak expression level of GADD45β. In resistant cell lines, pp53 and GADD45β levels decreased upon sorafenib exposure. In vivo, the combination treatment of sorafenib and 5-FU was equally effective as the respective monotherapy concerning tumor proliferation. Interestingly, treatment with either sorafenib or 5-FU resulted in a significant decrease of VEGFR1 and PDGFRβ expression intensity.

Conclusions

In colorectal cancer, a sensitivity towards sorafenib exists, which seems similarly effective as a 5-FU monotherapy. A combination therapy, in contrast, does not show any additional effect.

This study was presented at the 22nd EORTC-NCI-AACR Symposium on “Molecular Targets and Cancer Therapeutics,” 2010, in a poster presentation.

Introduction

Colorectal cancer ranges among the three most frequent malignancies in Western countries [1, 2]. Survival is determined by local recurrence, lymphatic, and hematogenous dissemination [3]. Due to improved therapeutic strategies, the overall survival in stage IV colorectal cancer has increased from 8 months to more than 2 years during the last decade [4‐6].

Besides new chemotherapeutic drugs, such as platinum derivates (oxaliplatin) and topoisomerase II inhibitors (irinotecan), the introduction of biologicals targeting tumor neovascularization or growth signaling significantly has improved response and prognosis [4‐6].

Specific mutations in tumor-suppressor genes (APC, DCC, p53) and oncogenes (K-ras) occur during the adenoma–carcinoma sequence of colorectal cancer [7‐9]. The K-ras mutation status was the first key to personalized therapy in colorectal cancer, as anti-EGFR strategies were shown to be effective in K-ras wild types only [10].

Receptor tyrosine kinases (RTKs) are transmembrane receptors containing extracellular ligand-binding domains connected to intracellular catalytic domains [11]. The growth factors VEGF/PDGF/EGF and their receptors VEGFR1-3, PDGFRα/β, and EGFR are critical in the process of (lymphatic) neo-angiogenesis and dissemination in human cancer [12‐16]. Inhibition of RTKs with sorafenib has been successful in renal and hepatocellular cancers [17, 18]. Two phase I studies revealed a disease stabilization in pretreated colorectal cancer patients receiving sorafenib in combination with either irinotecan or oxaliplatin [19, 20]. However, recent phase II/III studies testing other multi-tyrosine kinase inhibitors in human colorectal cancer have failed to show any benefit [21]. So far only one randomized Phase III study with Regorafenib improved survival times after failure of all approved standard therapies [22]. Therefore, the impact of combinational therapies (sorafenib + chemotherapy) remains controversial. Preclinical data as well as experimental data explaining interaction mechanisms are widely missing. Thus, we initiated this study to examine sorafenib targeted RTK expression and to analyze the in vivo effect of sorafenib alone or in combination with the classical chemotherapeutic backbone 5-fluorouracil (5-FU).

Material and methods

Cell culture

The human colorectal cancer cell lines SW480 [K-ras mt, B-Raf wt, PI3K wt, p53 mt], SW620 [K-ras mt, B-raf wt, PI3K wt, p53 mt], and HT29 [K-ras wt, B-raf mt, PI3K wt, p53 mt] were cultured in RPMI 1640 (Invitrogen, Germany) supplemented with 10 % FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex, Germany), and 1 mM l-glutamine (Invitrogen, Germany). The human colorectal cancer cell line Caco2 [K-ras wt, B-Raf wt, PI3K wt, p53 mt] was cultured in DMEM (Invitrogen, Germany) supplemented with 10 % FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex, Germany), and 1 mM l-glutamine (Invitrogen, Germany).

Proliferation assays and chemosensitvity

For proliferation assays, 5 × 10³ SW480, SW620, Caco2, or HT-29 cells were plated in 96-well plates and cultured as described above. Twelve hours after plating sorafenib (0, 5, and 10 μg/ml), 5-FU (0.5 mg/ml) ± sorafenib (5 μg/ml), irinotecan (1 mg/ml) ± sorafenib (5 μg/ml), or oxaliplatin (0.5 mg/ml) ± sorafenib (5 μg/ml) were added to the medium. The amount of cells per well was determined by luminescence assay (CellTiter-Glo Cell Viability assay, Promega, USA). Each condition was performed in quadruplicates.

For apoptosis analyses, 2 × 10⁵ cells were seeded per 6 wells, respectively. Twelve hours after plating, cells were treated for 24 h as mentioned above. Suspended cells were collected, and adherent cells were trypsinized prior to fixation with 100 % ethanol, staining with propidium iodide and analyzation by FACS without gating. Each condition was performed in quadruplicates.

Migration assay

SW480, SW620, Caco2, or HT29 cells (2 × 10⁶ ) were seeded per 6 wells, cultured for 24 h, serum-starved (2 % FCS only) for 12 h, and exposed to sorafenib at different concentrations (0, 5, or 10 μg/ml) for 6 h. Migration was assayed with 24-well HTS FluoroBlock Inserts in triplet approaches (8 μM pore size; Becton Dickinson, USA).

In brief, 4×10⁴ cells were resuspended in RPMI1640/DMEM medium containing 2 % FCS and 10 ng/ml CXCL12 and added to the upper chamber. Subsequently, RPMI1640/DMEM medium with 20 % FCS and 100 ng/ml CXCL12 (Sigma, Germany) was added to the lower chamber. Chambers were incubated for 24 h at 37 °C in a humid atmosphere of 5 % CO₂. After incubation, the amount of migrated cells in the lower chamber was determined by luminescence assay (CellTiter-Glo, Cell Viability assay, Promega, USA). Each condition was performed in triplicate.

Caspase assay

Cells were treated with placebo or sorafenib (5 and 10 μg/ml, respectively). After incubation for 16 h, cells were lysed in buffer containing 20 mM Tris/HCl pH 8.0, 5 mM EDTA, 0.5 % Triton X-100, and onefold complete protease inhibitor cocktail (Roche, Germany). Protein concentration was determined by Bradford assay (Sigma, Germany). Sixty micrograms of protein was incubated in reaction buffer (25 mM HEPES pH 7.5, 50 mM NaCl, 10 % glycerol, 0.05 % CHAPS, and 5 mM DTT) in the presence of 50 μM fluorogenic substrate (Biomol, Germany), which was specific for caspase 3 (DEVD-AMC); caspase 6, 8, and 10 (Ac-IETD-AFC); or caspase 9 (Ac-LEHD-AFC). Analyses were performed in triplicates.

Assays were performed in black micro-titer plates (Nunc, Germany), and after 1 h incubation at 37 °C, the generation of free AMC or AFC was measured using a fluorometer plate reader (Appliscan, Thermo Fisher, Germany) at an excitation wavelength of 380 nm (AMC and AFC) and an emission wavelength of 460 nm (AMC) or 505 nm (AFC).

Western blot analysis

SW480, SW620, Caco2, or HT29 cells (2 × 10⁶) were harvested after a 12-h-long exposition to placebo or sorafenib (5 and 10 μg/ml, respectively). Cells were washed twice with phosphate-buffered saline (PBS; 1×) and lysed in 2× RIPA solution. For Western blot analysis, 100 μg of protein was loaded on 8–12 % SDS-PAGE gels, respectively. After separation, the gel was transferred to a PVDF membrane (Roth, Karlsruhe, Germany). Proteins (AKT/pAKT, MEK/pMEK, PI3K/pPI3K, mTOR/pmTOR, P53/pp53, FoxO3a/pFoxO3a, GADD45β, and alpha tubulin) were detected with specific primary antibodies (Table 1; 4 °C, overnight). The specific secondary antibodies were exposed for 1 h at room temperature (Table 1). For visualisation, the Roti Lumin systems 1 and 2 were applied (P79 and P80, Roth, Karlsruhe, Germany). Each condition was performed in duplicates.

Table 1

Antibodies used for Western blotting

Antibody	Manufacturer	Order number	Secondary antibody	Size (kDa)	Dilution
Rabbit-anti-human pPI3K	Cell Signaling	4228	Goat-anti-rabbit IgG	85/60	1:1,000
Rabbit-anti-human PI3K	Cell Signaling	4257	Goat-anti-rabbit IgG	85	1:1,000
Rabbit-anti-human pAKT	Cell Signaling	9267	Goat-anti-rabbit IgG	60	1:1,000
Rabbit-anti-human AKT	Cell Signaling	4685	Goat-anti-rabbit IgG	60	1:1,000
Rabbit-anti-human pmTOR	Cell Signaling	2971	Goat-anti-rabbit IgG	289	1:1,000
Rabbit-anti-human mTOR	Cell Signaling	2983	Goat-anti-rabbit IgG	289	1:1,000
Rabbit-anti-human pMEK	Cell Signaling	9121	Goat-anti-rabbit IgG	45	1:1,000
Rabbit-anti-human MEK	Cell Signaling	9122	Goat-anti-rabbit IgG	45	1:1,000
Goat-anti-human GADD45β	Santa Cruz Biotechnology	sc-8776	Donkey-anti-goat IgG	18	1:500
Mouse-anti-human α-Tubulin	Sigma Aldrich	t5168	Goat-anti-mouse IgG	48,5	1:2,000
Goat-anti-mouse IgG	Santa Cruz Biotechnology	sc-2031	–	–	1:10,000
Goat-anti-rabbit IgG	Santa Cruz Biotechnology	sc-2030	–	–	1:10,000
Donkey-anti-goat IgG	Santa Cruz Biotechnology	sc-2033	–	–	1:10,000

Subcutaneous xenograft tumor system

HT29 tumor cells (1 × 10⁷) were suspended in 0.2 ml pure RPMI1640 medium and 1× PBS (1:1) and applied by subcutaneous injection into the left flank of 7–8-week-old female nod-SCID mice. Nod-SCID mice were irradiated with 1.8 Gy 1 day prior to s.c. injection of tumor cells. As soon as the tumors reached a size of 10 mm, animals received i.p. injections of placebo (group 1; 200 μl, 5 days/week; 25 % cremophor in NaCl 0,9 %), sorafenib (group 2; 200 μl; 5 days per week; 0.12 mg/dose solved in 25 % cremophor; 30 mg/kg/week), 5-FU (group 3; 200 μl; three times a week; 0.18 mg/dose solved in 25 % cremophor; 25 mg/kg/week) or sorafenib + 5-FU (group 4; 200 μl; combination of group 2 and 3). The size of tumors was measured manually twice weekly. Tumors grew for 4 weeks. Thereafter, tumor nodules were excised and measured manually with a vernier micrometer.

Immunohistochemistry

Excised tumors obtained from the experimental animals were paraffin-embedded. After obtaining adequate slides, the tissue samples were screened for Ki-67, PDGFA, VEGFA, VEGFR1, VEGFR2, PDFGRα, and PDGFRβ protein expression by immunohistochemistry. To that purpose, the tissues were deparaffinized, rehydrated, and subsequently incubated with the respective primary antibodies [anti-PDGFRα (sc-338); 1:200, 2 h, Santa Cruz Biotechnology, CA, USA; anti-PDGFRβ (3169), 1:40, 2 h, Cell Signaling Technology, MA, USA; PDGFA (NBP1-19781), 1:100, 2 h, Novus Biologicals, Cambridge, UK; VEGFA (ab46154), 1:200, 2 h, Abcam plc, Cambridge UK; VEGFR1 (RB-9049-R7), 1:50, 2 h, Thermo Fisher Scientific GmbH Neomarkers, Germany; VEGFR2 (RB-9239-R7), 1:50, 2 h, Thermo Fisher Scientific GmbH Neomarkers, Germany; VEGFR3 (sc-321), 1:200, 2 h, Santa Cruz Biotechnology, Germany; Ki-67 (mib1), 1:100, 2 h, Dako, Germany; Envision flex plus^TM, Autostainer, Dako, Germany]. The secondary antibody (anti-rabbit-mouse-goat antibody) was incubated for 15 min at room temperature, followed by incubation with streptavidin-POD (Dako, Germany) for 15 min. Antibody binding was visualized using AEC solution (Dako, Germany). Afterwards, the tissues were counterstained by haemalaun solution (Dako, Germany). The expression of the respective tyrosine kinase was evaluated using a scoring system. Expression strength of PDGFA, VEGFA, VEGFR1, VEGFR2, VEGFR3, PDFGRα, and PDGFRβ was classified as negative (0), low (1), medium, (2) and high (3). All slides were independently evaluated by three investigators. The Ki-67 expression was measured as percentage of Ki-67 expressing cells.

Statistics

In order to assess dependence of growth factor and Ki-67 expression on treatment with 5-FU and sorafenib, the minimum, the maximum, the median, and the quartiles in subgroups were calculated. For Ki-67 analyses, the mean and standard deviations were calculated and displayed in box plots. Ki-67 was measured three times for each specimen; averages were analyzed using two-way analysis of variance. To compare growth factor expression between treatment groups the Kruskal–Wallis tests was used, followed by pairwise Wilcoxon test if the Kruskal–Wallis test gave a p value ≤0.05.

All tests were performed with exploratory intention, associations with p values ≤0.05 might warrant further consideration. Statistical analysis was performed using SAS 9.3 2002–2010 by SAS Institute Inc., Cary, NC, USA.

Results

Proliferation assay

Inhibition of tumor growth through low dose sorafenib was seen in all cell lines, except for Caco2 (Table 2; Fig. 1a). High-dose sorafenib eradicated SW480 and HT29 cells significantly and SW620 cell less effectively, whereas Caco2 cells revealed a decelerated tumor cell growth only. Mutation status of K-ras, B-Raf, PI3K, or p53 did not correlate with resistance.

Table 2

Functional effects of sorafenib in distinct colorectal cancer cell lines

https://static-content.springer.com/image/art%3A10.1007%2Fs00384-012-1551-2/MediaObjects/384_2012_1551_Tab2_HTML.gif

Migration assay

The treatment with low-dose sorafenib (5 μg/ml) significantly inhibited migration only in SW480 and Caco2 cells (Table 2). High-dose (10 μg/ml) sorafenib nonsignificantly inhibited migration in HT29 and SW620 cells.

Caspases assays

Caspases 6, 8, and 10

Treatment with sorafenib significantly induced caspases 6, 8, and 10 activity in SW480 but not in HT29 cells (Table 2). In contrast, exposure to sorafenib significantly decreased caspases 6, 8, and 10 activity in Caco2 and SW620 cells

Caspase 9

Treatment with sorafenib significantly induced caspase 9 activity in SW480 but did not impact on HT29, Caco2, or SW620 cells.

Caspase 3

Treatment with sorafenib did not modify caspase 3 activity in SW480, HT29, or SW620 cells. However, caspase 3 activity was significantly decreased in Caco2 cells.

Chemosensitivity assay (apoptosis and proliferation)

5-Fluorouracil

Combining 5-FU and sorafenib revealed additive effects (proliferation and apoptosis) in SW480 and HT 29 cells but not in SW620 or Caco2 cells (Fig. 1b; Table 3). In contrast, the combination even increased proliferation of Caco2 cells.

Table 3

Effect of combining sorafenib with classical chemotherapeutic drugs

		SW480 (%)	p value	HT29 (%)	p value	Caco2 (%)	p value	SW620 (%)	p value
Proliferation
5-FU +	Placebo	150.3 ± 16.0	0.061	262.3 ± 25.9	0.004	337.2 ± 25.3	<0.001	97.2 ± 8.5	0.186
5-FU +	Sorafenib	127.7 ± 11.9		218.5 ± 20.2		468.2 ± 29.1		104.1 ± 8.5
Irinotecan +	Placebo	59.0 ± 7.0	0.002	217.7 ± 23.8	<0.001	585.1 ± 51.9	0.66	120.6 ± 9.7	<0.001
Irinotecan +	Sorafenib	39.1 ± 2.6		116.2 ± 13.1		570.4 ± 78.6		75.1 ± 8.1
Oxaliplatin +	Placebo	56.6 ± 3.5	0.1	204.7 ± 36.1	<0.001	308.4 ± 33.2	<0.001	10.2 ± 0.8	<0.001
Oxaliplatin +	Sorafenib	61.7 ± 6.6		114.8 ± 28.6		450.1 ± 20.0		13.7 ± 1.7
Apoptosis
5−FU		17.23 ± 2.67	0.002	5.28 ± 0.07	0.047	14.38 ± 1.15	0.32	5.84 ± 2.45	0.15
5−FU + sorafenib		42.27 ± 0.74		20.55 ± 1.62		16.98 ± 3.38		10.98 ± 1.73
Sorafenib		16.43 ± 1.45	<0.001	11.78 ± 1.56	0.031	17.12 ± 0.37	0.95	9.07 ± 4.65	0.66
Irinotecan		41.62 ± 4.12	0.004	24.08 ± 2.82	<0.001	12.38 ± 2.79	0.041	39.07 ± 3.01	0.238
Irinotecan + sorafenib		71.57 ± 1.25		56.23 ± 1.1		19.74 ± 3.2		26.44 ± 7.96
Sorafenib		16.43 ± 1.45	<0.001	11.78 ± 1.56	0.002	17.12 ± 0.37	0.29	9.07 ± 4.65	0.146
Oxaliplatin		39.26 ± 1.23	<0.001	24.93 ± 1.87	<0.001	38.52 ± 3.12	0.6	40.71 ± 1.31	0.058
Oxaliplatin + sorafenib		31.47 ± 1.15		46.92 ± 1.21		36.84 ± 2.13		30.62 ± 0.04
Sorafenib		16.43 ± 1.45	<0.001	11.78 ± 1.56 %	<0.001	17.12 ± 0.37	<0.001	9.07 ± 4.65	<0.001

Irinotecan

Combining 5-FU and irinotecan demonstrated clear additive effects in SW480, HT29 (proliferation and apoptosis), and SW620 (proliferation only) cells and even slightly increased apoptosis of Caco2 cells (Fig. 1c). However, no effect was seen in SW620 cells.

Oxaliplatin

Combining oxalipaltin and sorafenib revealed an additive effect (proliferation and apoptosis) in HT29 cells only but not in SW480, SW620, or Caco2 cells (Fig. 1d). In contrast, the combination increased proliferation of SW480, Caco2, and SW620 cells and inhibited apoptosis of SW480 and SW620 cells.

Signal cascade inhibition

In order to investigate the relevance of sorafenib in the inhibition of signal cascades, we analyzed diverse pathways (Fig. 2a). Upon exposure with increasing sorafenib doses, we observed an inhibition of the Ras–Raf pathway (pMEK) in SW620 but an induction in Caco2 cell lines. This pathway remained unchanged in SW480 and HT29 cells.

The AKT pathway was specifically altered in sensitive cell lines: SW480 and HT29 cells revealed only weak–absent pAKT, but AKT expression was significantly suppressed upon exposure with increasing sorafenib doses. In contrast, the resistant cell line Caco2 did not show any AKT suppressive behavior. An inhibition of pPI3K and pAKT was seen in Caco2 but not in SW620.

These results raise the question of whether a suppression of AKT expression correlates with responsiveness to sorafenib. As it has been previously reported that GADD45ß takes control when AKT is absent, we analyzed GADD 45-associated genes.

Potential resistance indicators

We observed that sorafenib-sensitive cell lines revealed almost absent pAKT, weak GADD45β, and medium–strong FoxO3a expression levels (Fig. 2b). In contrast, resistant cell lines showed medium pAKT, intensive GADD45β, and weak–medium FoxO3a expression levels. GADD45β expression levels discriminated best between sensitive and resistant cell lines.

Analyzing the impact of sorafenib on protein expression, we observed that the sensitive cell line SW480 reacts with a pp53 and a GADD45β upregulation upon increasing sorafenib doses. In contrast, resistant cell lines revealed primarily intense (Caco2, SW620) and, upon sorafenib exposure, decreasing (Caco2) GADD45β expression levels. Similarly, both resistant cell lines decreased pp53 levels upon exposure to increasing sorafenib doses.

In vivo xenograft model

Ex vivo analyses of tumor size indicated that—compared to placebo control—only a sorafenib monotherapy significantly decreased tumor size (220 ± 3.06 % versus 95.8 ± 4.34 %; P = <0.0001), while a 5-FU monotherapy inhibited tumor growth nonsignificantly (220 ± 3.06 % versus 124 ± 20.8 %; P = 0.097) (Fig. 3). The 5-FU plus sorafenib combination therapy was equipotent to 5-FU monotherapy (220 ± 3.06 % versus 146 ± 24.89 %; P = 0.085), but inferior compared to sorafenib monotherapy (P = 0.068), in this explorative analysis.

Therapeutic effect on Ki-67 proliferation index

Analysis of the proliferation index showed that sorafenib monotherapy and 5-FU monotherapy were equally effective in reducing the proliferation index as compared to placebo. However, the combination therapy of sorafenib and 5-FU did not result in further reduction of proliferation rates.

For Ki-67, analysis of variance demonstrated a significant effect for sorafenib (p = 0.0101), and for 5-FU + sorafenib interaction (p = 0.0049), the 5-FU main effect was borderline significant (p = 0.0537) as compared to the control group. On average, treatment with 5-FU lowered Ki-67 expression by 4.9 %, treatment with sorafenib on average lowered Ki-67 by 6.8 %. However, the effect was not additive: animals treated with both substances had higher Ki-67 values than animals treated with only one substance. The means for each treatment group are given in Fig. 4.

Therapeutic effect on cytokine expression

Specimens (N = 21) resected from 20 animals were included in the analysis. Each treatment group consisted of five animals, with six specimens available in the group treated with combined 5-FU and sorafenib (due to two tumors in one animal). The distribution of growth factor expression is given in Table 4. A summary of the Ki-67 measurements is given in Table 5.

Table 4

Summary measures for growth factor expression in treatment groups

Treated with 5-FU	Treated with sorafenib	Label	N	Minimum	Lower quartile	Median	Upper quartile	Maximum
No	No	VEGFR 1	5	2	2	2.5	2.5	2.5
		VEGFR 2	2	2	2	2	2	2
		VEGFR 3	5	1.5	2	2.5	3	3
		PDGFR alpha	5	0	0.5	0.5	1	1.5
		PDGFR beta	5	2	2	2	2	2
		VEGF A in cytoplasm	5	0.5	2	2.5	3	3
		VEGF A in nucleus	3	2	2	2	2	2
		PDGF A	5	0	1	1.5	1.5	2
	Yes	VEGFR 1	5	0.5	0.5	1	1	1
		VEGFR 2	5	0	0	0.5	1	1.5
		VEGFR 3	5	1.5	1.5	2	2.5	3
		PDGFR alpha	5	0	0.5	0.5	0.5	1.5
		PDGFR beta	5	0	0.5	0.5	0.5	1
		VEGF A in cytoplasm	5	1	1	1	1	2
		VEGF A in nucleus	5	0	0	0	1	2
		PDGF A	5	0	0	0	1	1.5
Yes	No	VEGFR 1	5	0.5	0.5	1	1	1.5
		VEGFR 2	5	0	0	0	0.5	1
		VEGFR 3	5	1	1.5	1.5	2	3
		PDGFR alpha	5	0	0	0.5	1.5	1.5
		PDGFR beta	5	0	0	0	1	1.5
		VEGF A in cytoplasm	5	0.5	1	1	2	2.5
		VEGF A in nucleus	5	0	0	1	2	3
		PDGF A	5	0	0	0	0	0.5
	Yes	VEGFR 1	6	1	1	1.3	1.5	1.5
		VEGFR 2	4	0	0	0.3	1	1.5
		VEGFR 3	6	1.5	2	2.3	2.5	3
		PDGFR alpha	6	0.5	0.5	1	1	1
		PDGFR beta	6	0	0	0.5	0.8	1
		VEGF A in cytoplasm	6	1	1	1	1	1.5
		VEGF A in nucleus	6	0	0	1	2	2
		PDGF A	6	0	0	0	1	1

Table 5

Summary of Ki-67 measurements

Treated with 5-FU	Treated with sorafenib	N	Mean	Standard deviation	Minimum	Lower quartile	Median	Upper quartile	Maximum
No	No	5	48.9	3.3	44.3	47	49.7	51.7	52
No	Yes	5	34.5	4.8	28	33	33.7	37.3	40.7
Yes	No	5	36.5	5.3	29.7	34.3	35	40	43.3
Yes	Yes	6	37.3	7	28.5	34	36.7	38	49.7

There were substantial differences in the expression of PDGFRβ (p = 0.002) and VEGFR1 (p = 0.0002) between the control group and the different treatment groups. There was also a tendency for different expression of PDGFA (p = 0.0799) between different groups (Fig. 5a). Representative tissue examples are shown in Fig. 5b.

The highest expression levels of VEGFR1 were seen in the untreated control group and differed in relation to the group treated with 5-FU (p = 0.0079), sorafenib (p = 0.0079), and 5-FU + sorafenib (p = 0.0022). The difference among treatment groups remained within random variation.

A similar result was seen for PDGFRβ expression. The untreated control group showed the highest expression level differing from all treatment groups (p = 0.0079 when compared with 5-FU or sorafenib, p = 0.0043 when compared to 5-FU + sorafenib).

The tentative differences in PDGFA expression resulted from higher values in the untreated control group as compared to lower values in the treated groups.

The other growth factors did not show any differences beyond random variation(p > 0.1). The p values in the Kruskal–Wallis test were: PDGFRα, p = 0.8355; VEGFR2, p = 0.1020; VEGFR 3, p = 0.5058; cytoplasmic VEGF A, p = 0.2701; and nuclear VEGF A, p = 0.3397.

Discussion

The approach of inhibiting RTKs with sorafenib has been successful in renal and hepatocellular cancers [17, 18]. A phase I study revealed disease stabilization in pretreated colorectal cancer patients [20]. Except of one recent study with Regorafenib, recent phase II/III studies testing other multi-tyrosine kinase inhibitors in colorectal cancer failed to show any benefit [21, 22]. So far, no molecular markers have been identified which are helpful in stratifying the patients.

We performed defined functional in vitro analyses in order to identify sorafenib-sensitive and sorafenib-resistant cell lines. While HT29 and SW480 were found to be sorafenib sensible, Caco2 was resistant and SW620 showed features of resistance. However, the mutation status of K-ras, B-Raf, PI3K, or p53 did not correlate with resistance.

Combining sorafenib with chemotherapeutic drugs used in colorectal cancer revealed an additive effect in growth inhibition and apoptosis induction in SW480 (except for oxaliplatin) and HT29 cells, whereas in Caco2 cells, apoptosis was not increased and proliferation even stimulated (5-FU or oxaliplatin). These data are in line with previous reports describing a reduced cellular uptake of oxaliplatin and generation of DNA adducts in specific colorectal cancer cells through sorafenib [23]. Thus, combination with oxaliplatin seems disadvantageous in specific settings. The effect of sorafenib on migration was marginal and of no significant importance.

Induction of apoptosis might explain the different observations made upon sorafenib exposure: While activity of caspases 6, 8, and 10 was induced in sensitive SW480 cells, it was decreased in the resistant cell lines. Furthermore, SW480 reacted with an increased activity of caspase 9. In contrast, activity of caspase 3 was decreased in Caco2 cells upon exposure to sorafenib. An induction of caspase 3 activity, as seen in prostate cancer cells, was not observed in colorectal cancer cells [24]. Our data reveal that resistance to sorafenib is associated with inhibition of specific pro-apoptotic pathways. However, sorafenib is also known to induce caspase-independent apoptosis, mediated through nuclear translocation of AIF [25].

We observed an inhibition of the Ras–Raf pathway (pMEK) in SW620 cell lines only, matching sorafenib’s function as a Raf inhibitor [25]. While sensitive cell lines revealed only a weak–absent pAKT expression, AKT expression was clearly suppressed upon exposure with increasing sorafenib doses. In contrast, the resistant cell line Caco2 did not show such AKT suppressive behavior. These observations match a previous report that a constitutively active AKT protects cells against sorafenib/bortezomib-induced apoptosis [26, 27].

Sorafenib-sensitive cells lines were defined by almost absent pAKT, medium–strong FoxO3a, and hint GADD45β levels. The tumor suppressor FoxO3 belongs to a subclass of the forkhead transcription factors, being inhibited by activation of the PI3K pathway. Downregulation of FoxO3 is thus considered a consequence of pAKT activity.

In contrast, resistant cell lines showed medium pAKT, weak FoxO3a, and very intense GADD45β levels. GADD45β expression levels discriminated best between sensitive and resistant cell lines. GADD45 is a stress sensor modulating the response of cells to genotoxic or oxidative stress [28‐30]. In specific colon cancer cells, GADD45β over-expression was linked to protection from platin induced death, matching our observations [31]. Being an apoptosis modulator, activation of GADD45β prevents the propagation of damaged cells, causing an arrest in cell growth and apoptosis after exposure to toxins [32]. This regulation seems intact in SW480 cells but reversed in resistant cells; GADD45ß was downregulated in Caco2 upon sorafenib treatment, going along with a sorafenib-mediated inhibition of caspases 6, 8, and 10. As a downstream effector of p53, GADD45β was confirmed to be specifically downregulated in HCC, which was associated to the extent of p53 mutation [33]. We observed a pp53 and a GADD45β upregulation in some sensitive cell line (SW480) upon exposure to sorafenib. In contrast, resistant cell lines showed primarily high (Caco2, SW620) and, upon sorafenib exposure, decreasing (Caco2) GADD45β and pp53 levels. These data are in line with observations in HCC, in which GADD45β induction by sorafenib occurred only in sensitive hepatocellular carcinoma cell lines, independent of the Raf/MEK/ERK signaling pathway [34].These findings confirm our definition of sensitive cell lines, in which sorafenib induces apoptosis and inhibits proliferation.

In vivo, Wilhelm and colleagues described a potent growth inhibition of HT29 xenografts at sorafenib doses of 7.5 mg/kg. We studied four different groups in vivo: placebo, 5-FU, sorafenib, and 5-FU + sorafenib. 5-FU was chosen, being the backbone of most chemotherapeutic protocols in colorectal cancer. Sorafenib was applied at 5 mg/kg, matching 400 mg/day as used in combination therapies [19, 20].

Interestingly, we observed that a sorafenib monotherapy was at least equally effective as the 5-FU monotherapy or as the combination therapy and even tended to inhibit in vivo tumor growth somewhat better than the combination therapy.

The proliferation index was significantly reduced in all treatment groups as compared to the control group but displayed similar results for mono-agent therapy and the combination therapy. Since only small numbers were analyzed, a possibility exists that larger treatment groups might demonstrate even more distinct differences. However, we clearly demonstrate that combination of sorafenib and chemotherapy did not result in any additive effects. In contrast, it seems that treatment effects are partially cancelled when 5-FU and sorafenib are applied simultaneously.

Expression rates of receptor tyrosine kinases VEGFR1 and PDGFRβ as well as of the ligand PDGFA were decreased by all treatment regimens used. However, no significant differences were detected between treatment groups.

Inhibition of receptor tyrosine kinases through sorafenib could potentially lead to a selection of low target expressing tumor cells. Combination regimens of sorafenib and 5-FU might reduce sorafenib target expression leading to a similar proliferation effect as under 5-FU monotherapy. However, the adverse events in humans might rather be additive. Our results indicate that there is no additive effect in combination of these two treatment mechanisms and that combination might only add adverse events. Therefore, in future studies preferentially sorafenib monotherapy versus sequential treatment regimens (inductiontherapy via chemotherapy–maintenance via sorafenib) should be explored.

Conclusion

Diverse tyrosine kinase inhibitors have failed in colorectal cancer. However, sorafenib still seems promising in distinct settings, if applied as monotherapy. In our human colon cancer xenograft model, it seems that treatment effects are partially cancelled when 5-FU and sorafenib are applied simultaneously. However, monotherapy with sorafenib seems to be sufficient for tumor control in our human colon cancer xenograft model, especially considering the application advantage and toxicity profile.

Acknowledgments

This study was financially supported by the Bayer Healthcare AG.

Disclaimer

Carl Schimanski received research funding; Markus Moehler, speech honoraria; and Peter R. Galle, consulting and speech honoraria.

Open Access

This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Unsere Produktempfehlungen

Die Chirurgie

Print-Titel

Das Abo mit mehr Tiefe

Mit der Zeitschrift Die Chirurgie erhalten Sie zusätzlich Online-Zugriff auf weitere 43 chirurgische Fachzeitschriften, CME-Fortbildungen, Webinare, Vorbereitungskursen zur Facharztprüfung und die digitale Enzyklopädie e.Medpedia.

Bis 30. April 2024 bestellen und im ersten Jahr nur 199 € zahlen!

Jetzt informieren

e.Med Interdisziplinär

Kombi-Abonnement

Für Ihren Erfolg in Klinik und Praxis - Die beste Hilfe in Ihrem Arbeitsalltag

Mit e.Med Interdisziplinär erhalten Sie Zugang zu allen CME-Fortbildungen und Fachzeitschriften auf SpringerMedizin.de.

Jetzt testen ¹

e.Med Innere Medizin

Kombi-Abonnement

Mit e.Med Innere Medizin erhalten Sie Zugang zu CME-Fortbildungen des Fachgebietes Innere Medizin, den Premium-Inhalten der internistischen Fachzeitschriften, inklusive einer gedruckten internistischen Zeitschrift Ihrer Wahl.

Jetzt testen ²

Greenlee RT, Murray T, Bolden S, Wingo PA et al (2000) Cancer statistics, 2000. CA Cancer J Clin 50:7–33PubMedCrossRef

Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, Jemal A, Ward E, Anderson RN, Edwards BK (2003) Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control. J Natl Cancer Inst 95:1276–1299PubMedCrossRef

August DA, Ottow RT, Sugarbaker PH (1984) Clinical perspective of human colorectal cancer metastasis. Cancer Metastasis Rev 3:303–324PubMedCrossRef

Douillard JY, Siena S, Cassidy J, Tabernero J, Burkes R, Barugel M, Humblet Y, Bodoky G, Cunningham D, Jassem J, Rivera F, Kocakova I et al (2010) Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol 28(31):4697–7705PubMedCrossRef

Sobrero A, Ackland S, Clarke S, Perez-Carrion R, Chiara S, Gapski J, Mainwaring P, Langer B, Young S (2009) Phase IV study of bevacizumab in combination with infusional fluorouracil, leucovorin and irinotecan (FOLFIRI) in first-line metastatic colorectal cancer. Oncology 77:113–119PubMedCrossRef

Welch S, Spithoff K, Rumble RB, Maroun J (2010) Bevacizumab combined with chemotherapy for patients with advanced colorectal cancer: a systematic review. Ann Oncol 21(6):1152–1162PubMedCrossRef

Fearon ER, Vogelstein B (1990) A genetic model for colorectal tumorigenesis. Cell 61:759–767PubMedCrossRef

Cho KR, Vogelstein B (1992) Genetic alterations in the adenoma–carcinoma sequence. Cancer 70:1727–1731PubMedCrossRef

Vogelstein B, Kinzler KW (1993) The multistep nature of cancer. Trends Genet 9:138–141PubMedCrossRef

10.

Khambata-Ford S, Garrett CR, Meropol NJ, Basik M, Harbison CT, Wu S, Wong TW, Huang X, Takimoto CH, Godwin AK, Tan BR, Krishnamurthi SS et al (2007) Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol 25:3230–3237PubMedCrossRef

11.

Li E, Hristova K (2006) Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies. Biochemistry 45:6241–6251PubMedCrossRef

12.

Mobius C, Stein HJ, Becker I, Feith M, Theisen J, Gais P, Jutting U, Siewert JR (2003) The ‘angiogenic switch’ in the progression from Barrett’s metaplasia to esophageal adenocarcinoma. Eur J Surg Oncol 29:890–894PubMedCrossRef

13.

Liu XE, Sun XD, Wu JM (2004) Expression and significance of VEGF-C and FLT-4 in gastric cancer. World J Gastroenterol 10:352–355PubMed

14.

Yonemura Y, Endo Y, Tabata K, Kawamura T, Yun HY, Bandou E, Sasaki T, Miura M (2005) Role of VEGF-C and VEGF-D in lymphangiogenesis in gastric cancer. Int J Clin Oncol 10:318–327PubMedCrossRef

15.

Takahashi Y, Cleary KR, Mai M, Kitadai Y, Bucana CD, Ellis LM (1996) Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer. Clin Cancer Res 2:1679–1684PubMed

16.

Wang KL, Wu TT, Choi IS, Wang H, Reseetkova E, Correa AM, Hofstetter WL, Swisher SG, Ajani JA, Rashid A, Albarracin CT (2007) Expression of epidermal growth factor receptor in esophageal and esophagogastric junction adenocarcinomas: association with poor outcome. Cancer 109:658–667PubMedCrossRef

17.

Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C et al (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378–390PubMedCrossRef

18.

Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Staehler M, Negrier S, Chevreau C, Desai AA, Rolland F, Demkow T, Hutson TE et al (2009) Sorafenib for treatment of renal cell carcinoma: final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol 27:3312–3318PubMedCrossRef

19.

Mross K, Steinbild S, Baas F, Gmehling D, Radtke M, Voliotis D, Brendel E, Christensen O, Unger C (2007) Results from an in vitro and a clinical/pharmacological phase I study with the combination irinotecan and sorafenib. Eur J Cancer 43:55–63PubMedCrossRef

20.

Kupsch P, Henning BF, Passarge K, Richly H, Wiesemann K, Hilger RA, Scheulen ME, Christensen O, Brendel E, Schwartz B, Hofstra E, Voigtmann R et al (2005) Results of a phase I trial of sorafenib (BAY 43-9006) in combination with oxaliplatin in patients with refractory solid tumors, including colorectal cancer. Clin Colorectal Cancer 5:188–196PubMedCrossRef

21.

Saltz LB, Rosen LS, Marshall JL, Belt RJ, Hurwitz HI, Eckhardt SG, Bergsland EK, Haller DG, Lockhart AC, Rocha Lima CM, Huang X, DePrimo SE et al (2007) Phase II trial of sunitinib in patients with metastatic colorectal cancer after failure of standard therapy. J Clin Oncol 25:4793–4799PubMedCrossRef

22.

Cuteem EV, Sobrero AF, Siena S, Falcone A, Ychou M, Humblet Y, Bouche O, Mineur L, Barone C, Adenis A, Tabernero J, Yoshino T et al (2012) Phase III CORRECT trial of regorafenib in metastatic colorectal cancer (mCRC). J Clin Oncol 30(suppl; abstr 3502)

23.

Heim M, Scharifi M, Zisowsky J, Jaehde U, Voliotis D, Seeber S, Strumberg D (2005) The Raf kinase inhibitor BAY 43-9006 reduces cellular uptake of platinum compounds and cytotoxicity in human colorectal carcinoma cell lines. Anticancer Drugs 16:129–136PubMedCrossRef

24.

Huang R, Chen XQ, Huang Y, Chen N, Zeng H (2010) The multikinase inhibitor sorafenib induces caspase-dependent apoptosis in PC-3 prostate cancer cells. Asian J Androl 12:527–534PubMedCrossRef

25.

Panka DJ, Wang W, Atkins MB, Mier JW (2006) The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells. Cancer Res 66:1611–1619PubMedCrossRef

26.

Yu C, Friday BB, Lai JP, Yang L, Sarkaria J, Kay NE, Carter CA, Roberts LR, Kaufmann SH, Adjei AA (2006) Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways. Mol Cancer Ther 5:2378–2387PubMedCrossRef

27.

Chen KF, Yu HC, Liu TH, Lee SS, Chen PJ, Cheng AL (2010) Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation. J Hepatol 52:88–95PubMedCrossRef

28.

Kastan MB, Zhan Q, el-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ Jr (1992) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71:587–597PubMedCrossRef

29.

Liebermann DA, Hoffman B (2002) Myeloid differentiation (MyD)/growth arrest DNA damage (GADD) genes in tumor suppression, immunity and inflammation. Leukemia 16:527–541PubMedCrossRef

30.

Nemoto S, Finkel T (2002) Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science 295:2450–2452PubMedCrossRef

31.

Smith ML, Kontny HU, Bortnick R, Fornace AJ Jr (1997) The p53-regulated cyclin G gene promotes cell growth: p53 downstream effectors cyclin G and Gadd45 exert different effects on cisplatin chemosensitivity. Exp Cell Res 230:61–68PubMedCrossRef

32.

Zhang W, Bae I, Krishnaraju K, Azam N, Fan W, Smith K, Hoffman B, Liebermann DA (1999) CR6: A third member in the MyD118 and Gadd45 gene family which functions in negative growth control. Oncogene 18:4899–4907PubMedCrossRef

33.

Qiu W, David D, Zhou B, Chu PG, Zhang B, Wu M, Xiao J, Han T, Zhu Z, Wang T, Liu X, Lopez R et al (2003) Down-regulation of growth arrest DNA damage-inducible gene 45beta expression is associated with human hepatocellular carcinoma. Am J Pathol 162:1961–1974PubMedCrossRef

34.

Ou DL, Shen YC, Yu SL, Chen KF, Yeh PY, Fan HH, Feng WC, Wang CT, Lin LI, Hsu C, Cheng AL (2010) Induction of DNA damage-inducible gene GADD45{beta} contributes to sorafenib-induced apoptosis in hepatocellular carcinoma cells. Cancer Res 70:9309–9318PubMedCrossRef

Titel: Single-agent therapy with sorafenib or 5-FU is equally effective in human colorectal cancer xenograft—no benefit of combination therapy
verfasst von: Thomas C. Wehler
Swaantje Hamdi
Annett Maderer
Claudine Graf
Ines Gockel
Irene Schmidtmann
Michael Hainz
Martin R. Berger
Matthias Theobald
Peter R. Galle
Markus Moehler
Carl C. Schimanski
Publikationsdatum: 01.03.2013
Verlag: Springer-Verlag
Erschienen in: International Journal of Colorectal Disease / Ausgabe 3/2013
Print ISSN: 0179-1958
Elektronische ISSN: 1432-1262
DOI: https://doi.org/10.1007/s00384-012-1551-2

Neu im Fachgebiet Chirurgie

Vorsicht, erhöhte Blutungsgefahr nach PCI!

10.05.2024 Koronare Herzerkrankung Nachrichten

Nach PCI besteht ein erhöhtes Blutungsrisiko, wenn die Behandelten eine verminderte linksventrikuläre Ejektionsfraktion aufweisen. Das Risiko ist umso höher, je stärker die Pumpfunktion eingeschränkt ist.

Darf man die Behandlung eines Neonazis ablehnen?

08.05.2024 Gesellschaft Nachrichten

In einer Leseranfrage in der Zeitschrift Journal of the American Academy of Dermatology möchte ein anonymer Dermatologe bzw. eine anonyme Dermatologin wissen, ob er oder sie einen Patienten behandeln muss, der eine rassistische Tätowierung trägt.

Deutlich weniger Infektionen: Wundprotektoren schützen!

08.05.2024 Postoperative Wundinfektion Nachrichten

Der Einsatz von Wundprotektoren bei offenen Eingriffen am unteren Gastrointestinaltrakt schützt vor Infektionen im Op.-Gebiet – und dient darüber hinaus der besseren Sicht. Das bestätigt mit großer Robustheit eine randomisierte Studie im Fachblatt JAMA Surgery.

Chirurginnen und Chirurgen sind stark suizidgefährdet

07.05.2024 Suizid Nachrichten

Der belastende Arbeitsalltag wirkt sich negativ auf die psychische Gesundheit der Angehörigen ärztlicher Berufsgruppen aus. Chirurginnen und Chirurgen bilden da keine Ausnahme, im Gegenteil.

Update Chirurgie

Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.

Newsletter bestellen

Aktuelle BDC Leitlinien-Webinare

S3-Leitlinie „Diagnostik und Therapie des Karpaltunnelsyndroms“

Karpaltunnelsyndrom BDC Leitlinien Webinare

CME: 2 Punkte

Das Karpaltunnelsyndrom ist die häufigste Kompressionsneuropathie peripherer Nerven. Obwohl die Anamnese mit dem nächtlichen Einschlafen der Hand (Brachialgia parästhetica nocturna) sehr typisch ist, ist eine klinisch-neurologische Untersuchung und Elektroneurografie in manchen Fällen auch eine Neurosonografie erforderlich. Im Anfangsstadium sind konservative Maßnahmen (Handgelenksschiene, Ergotherapie) empfehlenswert. Bei nicht Ansprechen der konservativen Therapie oder Auftreten von neurologischen Ausfällen ist eine Dekompression des N. medianus am Karpaltunnel indiziert.

Prof. Dr. med. Gregor Antoniadis

S2e-Leitlinie „Distale Radiusfraktur“

Radiusfraktur BDC Leitlinien Webinare

CME: 2 Punkte

Das Webinar beschäftigt sich mit Fragen und Antworten zu Diagnostik und Klassifikation sowie Möglichkeiten des Ausschlusses von Zusatzverletzungen. Die Referenten erläutern, welche Frakturen konservativ behandelt werden können und wie. Das Webinar beantwortet die Frage nach aktuellen operativen Therapiekonzepten: Welcher Zugang, welches Osteosynthesematerial? Auf was muss bei der Nachbehandlung der distalen Radiusfraktur geachtet werden?

PD Dr. med. Oliver Pieske

Dr. med. Benjamin Meyknecht

S1-Leitlinie „Empfehlungen zur Therapie der akuten Appendizitis bei Erwachsenen“

Appendizitis BDC Leitlinien Webinare

CME: 2 Punkte

Inhalte des Webinars zur S1-Leitlinie „Empfehlungen zur Therapie der akuten Appendizitis bei Erwachsenen“ sind die Darstellung des Projektes und des Erstellungswegs zur S1-Leitlinie, die Erläuterung der klinischen Relevanz der Klassifikation EAES 2015, die wissenschaftliche Begründung der wichtigsten Empfehlungen und die Darstellung stadiengerechter Therapieoptionen.

Dr. med. Mihailo Andric

Die Highlights vom Kongress des American College of Cardiology 2024

Springer Medizin

Abstract

Background

Methods

Results

Conclusions

Introduction

Material and methods

Cell culture

Proliferation assays and chemosensitvity

Migration assay

Caspase assay

Western blot analysis

Subcutaneous xenograft tumor system

Immunohistochemistry

Statistics

Results

Proliferation assay

Migration assay

Caspases assays

Chemosensitivity assay (apoptosis and proliferation)

Signal cascade inhibition

Potential resistance indicators

In vivo xenograft model

Discussion

Conclusion

Acknowledgments

Disclaimer

Open Access

Unsere Produktempfehlungen

Die Chirurgie

e.Med Interdisziplinär

e.Med Innere Medizin

Weitere Artikel der Ausgabe 3/2013

Higher rate of perineural invasion in stent–laparoscopic approach in comparison to emergent open resection for obstructing left-sided colon cancer

Rapid morphological changes and loss of collagen following experimental acute colonic obstruction

Letter to the editor: a meta-analysis of MTHFR C677T polymorphism and colorectal cancer risk in East Asians

Predictive value of vascular endothelial growth factor overexpression in early relapse of colorectal cancer patients after curative resection

TNF-α-308 G>A polymorphism and colorectal cancer risk: a meta-analysis

IBS and the role of otilonium bromide

Update Chirurgie