Background
Osteosarcoma (OS) is the most common primary malignant tumor of the bone in children and adolescents [
1]. Great progress has been made in the therapy of OS due to the utilization of neoadjuvant chemotherapy and radiotherapy in combination with surgical resection. Overall survival has increased to 60–75% and has remained the same for the last two decades [
2]. Unfortunately, the prognosis of OS with metastasis is still poor; only 30% patients with metastatic OS achieve 5-year tumor-free survival [
3].
Cisplatin (cis-diamminedichloroplatinum II, DDP) is a common and effective chemotherapeutic drug used in the treatment of various human solid tumors, including bladder cancer, cervical cancer, small cell lung cancer and gastric cancer [
4]. DDP treatment is considered a useful chemotherapeutic method for preoperative induction therapy for OS with an improved survival rate [
5]. The general mechanism by which DDP kills cancer cells has been elucidated. Briefly, DDP induces DNA intrastrand cross links between adjacent purines, which results in DNA damage that leads to the inhibition of tumor cell invasion and initiation of apoptosis, or programmed cell death [
6]. DDP has an obvious killing effect on osteosarcoma cells; however, the toxicity and acquisition of intrinsic resistance by OS cells after long-term application of DDP remain major obstacles [
7]. Recently, some novel compounds, such as platinum complexes and vanadium complexes, have been developed that exhibit efficacy against human OS cell lines and even some chemoresistant OS cell lines. These compounds may represent a new class of potent anti-OS agents but had limited efficacy under experimentally controlled conditions. Furthermore, the putative mechanisms and biosafety of these novel compounds still need to be elucidated in future research [
8‐
10]. Therefore, there is an urgent need to develop a more effective and safe treatment strategy that combines a low dosage of DDP, one of the gold standard drugs in OS treatment, with other agents to decrease DDP-related side effects and chemoresistance.
Phytochemicals are a series of compounds that are extracted and purified from plants such as vegetables, fruits, spices, and grains. Many studies have demonstrated the pharmacological activities of phytochemicals, including antioxidant [
11], antimicrobial [
12], antidiabetic [
13], and anti-inflammatory effects [
14]. Most recently, the anticancer and chemoprevention properties of phytochemicals have attracted increasing interest from oncology researchers due to their low intrinsic toxicity in normal cells but prominent effects in cancerous cells [
15]. Phytochemicals can exhibit diverse inhibitory effects on the initiation, promotion, progression, invasion and metastasis of cancer [
16,
17]. Recent studies have shown that phytochemicals can restore the sensitivity of cancer cells to conventional chemotherapeutic drugs [
18]. Synergistic or additional effects of combinations of DDP and phytochemical compounds in cancer cells with acceptable side effects have also been demonstrated [
19,
20]. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide, CAP) is one of the major pungent ingredients of red pepper and has been widely used in clinical medicine for the treatment of pain and inflammation caused by various diseases [
21]. In addition, numerous studies and animal experiments have demonstrated the anticancer and chemopreventive properties of CAP [
22]. Our previous study showed that CAP has profound in vitro and in vivo antiproliferative effects against human OS cells. However, apoptotic effects in OS cells were only observed upon treatment with a relatively high concentration of CAP [
23]. Thus, we hypothesized that the combination of subtoxic concentrations of the phytochemical CAP and the conventional chemotherapeutic DDP could exhibit significant killing effects in OS cells. Here, we demonstrated that CAP synergistically potentiates the anticancer activity of DDP in OS cells in vitro and that the combination of CAP and DDP inhibits tumor growth more significantly than the control or any other single-administration group in the OS xenograft model. The possible molecular pathway underlying this effectiveness is also discussed. The present results suggest that CAP could serve as a candidate for further development as a chemotherapy adjuvant for the treatment of OS.
Methods
Reagents and chemicals
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin (DDP) was purchased from Qilu Pharm (Jinan, China). Fetal bovine serum (FBS) and Dulbecco’s-modified Eagle’s medium (DMEM) were purchased from HyClone (Logan, UT, USA). The cell viability and cytotoxicity test kit, namely Cell Counting Kit-8 (CCK-8), was purchased from Dojindo Molecular Technologies (Kimamoto, Japan). The Annexin V-FITC/propidium iodide (PI) double-staining test kit was purchased from KeyGen Biotech (Nanjing, China). RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF), the bicinchoninic acid (BCA) protein assay kit, bovine serum albumin (BSA), a JC-1 mitochondrial membrane potential assay kit (C2006), crystal violet, N-acetyl-L-cysteine (NAC) (S0077), the Reactive Oxygen Species Assay Kit (S0033) and mouse anti-human GAPDH (AG019–1) were purchased from Beyotime Biotech (Shanghai, China). Monodansylcadaverine (MDC) (G0170) was purchased from Solarbio (Beijing China). Bafilomycin A1 (Baf-A1) (S1413) was purchased from Selleck (Houston, USA). Rabbit anti-human Bax (cat. D2E11), rabbit anti-human Bcl-2 (D55G8) (cat. 4223), rabbit anti-human cytochrome c (cat. 11940), rabbit anti-human cleaved caspase-3 (cat. 9664), rabbit anti-human MMP-9 (cat. 13667), the Cell Cycle Regulation Antibody Sampler Kit (cat. 9932), the Autophagy Antibody Sampler Kit (cat. 4445), rabbit anti-human SQSTM1/p62 (cat. 8025), rabbit anti-human AKT (cat. 4691), rabbit anti-human phospho-AKT (Ser 473) (cat. 4060), rabbit anti-human mTOR (cat. 2983), rabbit anti-human phospho-mTOR (Ser 2448) (cat. 5536), rabbit anti-human SAPK/JNK(cat. 9252) and rabbit anti-human phospho-SAPK/JNK (Thr183/Tyr185) (cat. 4668) were purchased from Cell Signaling Technology (Boston, MA, USA). Rabbit anti-human PCNA (3888) and rabbit anti-human Ki67 (4381) were purchased from Boster Biological Technology (Wuhan China). Rabbit anti-human Survivin (cat. 7642) and rabbit anti-mouse MMP2 (cat. 86607) were purchased from Abcam (MA, USA). Goat anti-rabbit (cat. 111–035-003) and goat anti-mouse secondary antibodies (cat. 115–035-003) were purchased from Jackson ImmunoResearch Laboratories (PA, USA).
Cell culture
The OS cell lines MG63, 143B and HOS were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS, 100 u/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C.
Cell viability assay
A CCK-8 assay was used to determine the viability of cells after the different treatments. Briefly, OS cells were seeded into 96-well plates at a density of 5,000 cells/well and incubated overnight for adherence. To determine the individual effects of CAP and DDP on OS cells, cells were incubated with various concentrations of CAP (0, 50, 100, 150, 200, 250 or 300 μM) or DDP (0, 8.3, 16.7, 25, 33.3, 50, 66.7, 83.3, 100, 116.6 or 133.3 μM) for 24 h. To determine the combined effects of CAP and DDP, OS cells were exposed to various combinations of relatively low concentrations of CAP (50, 100 and 100 μM) and various concentrations of DDP (0, 16.7, 33.3, 50, 66.7 μM) for 24 h. To assess the change in viability after the use of inhibitors, OS cells were preincubated with inhibitors (Baf-A1 or NAC) for 1 h before exposure to vehicle, CAP, DDP or CAP/DDP combination. Following the different treatments, 10 μL of CCK-8 was added to each well, and the plates were incubated for an additional hour. The plates with cells were subsequently placed in a microplate reader to detect the absorbance at 450 nm. Cell viability was calculated using the following formula: cell viability (%) = experimental group absorbance value / control group absorbance value X 100%.
Combination index
The combined effect of DDP and CAP on OS cells was evaluated using the combination index (CI) as described previously [
24]. The combined effect is classified as follows: CI < 1 is a synergistic effect; CI = 1 is an additive effect; and CI > 1 is an antagonistic effect. CI analysis was performed using Calcusyn Graphing Software (Biosoft, Inc., MO, USA).
Cell apoptosis assay
Apoptosis was determined by flow cytometry using Annexin V-FITC/PI staining. Briefly, cells were seeded into 6-well plates (105 cells/well) and incubated overnight for adherence, and then the cells were exposed to CAP (100 μM), DDP (16.7 μM), or CAP and DDP combined for 24 h. After treatment, the cells were collected, washed twice with ice-cold phosphate-buffered saline (PBS), and stained with Annexin V-FITC and PI according to the manufacturer’s guidelines. The samples were then read on a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The distribution of viable (FITC-/PI-), early apoptotic (FITC+/ PI-), late apoptotic (FITC+/PI+) and necrotic (FITC-/PI+) cells was analyzed. Both early and late apoptotic cells were recorded as apoptotic cells, and the results are expressed as the percentage of total cells.
Mitochondrial membrane potential assay
The mitochondrial membrane potential (Δψm) was detected using a JC-1 assay kit. Cells were seeded into 6-well plates at a density of 105 cells/well and incubated overnight. After adherence, the cells were incubated with CAP (100 μM) or DDP (16.7 μM) alone or in combination for 24 h. Next, the cells were washed with PBS and incubated in medium containing 2 mM JC-1 at 37 °C for 20 min. After washing with ice-cold JC-1 buffer, the cells were directly observed under a fluorescence microscope. For flow cytometry assessments, after exposure to different treatments for 24 h, the cells were trypsinized, collected in medium containing JC-1 and then incubated at 37 °C for 20 min. After washing with ice-cold JC-1 buffer, the cells were analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
Cell cycle assay
The cell cycle distribution was analyzed by flow cytometry with PI staining. Briefly, cells were seeded into 6-well plates at a density of 105 cells/well and incubated overnight to allow adherence. Then, the cells were treated with CAP (100 μM), DDP (16.7 μM) or CAP in combination with DDP for another 24 h. The cells were trypsinized, collected and fixed in 70% ice-cold ethanol at − 20 °C overnight. Then, the cells were incubated with 10 mg/mL RNase and 50 μg/mL PI for 30 min. The cell cycle distribution was assessed using flow cytometry (BD Bioscience, Franklin Lakes, New Jersey).
Cell invasion assay
The in vitro invasion activities of OS cells were evaluated using Transwell chambers with a 0.22-μm pore size (Corning, Inc.). The chambers was washed with serum-free medium, and then a layer of Matrigel was evenly plated on the surface of the membrane. Cells (105 cells) were seeded into the upper chambers with 200 μL of treatment medium (control group, 100 μM CAP group, 16.7 μM DDP group and CAP/DDP combination group) and incubated in 24-well plates with 500 μL of medium containing 10% FBS in the lower chamber. After 24 h of treatment, non-invading cells were removed from the upper surfaces of the membranes. Invaded cells were stained with 1% crystal violet, and the cell numbers were counted under a microscope.
Gelatin zymography assay
Cells were seeded into 6-well plates at a density of 105 cells/well and incubated overnight to allow adherence. Then, the cells were treated with CAP (100 μM, serum-free medium), DDP (16.7 μM, serum-free medium), or CAP in combination with DDP for another 24 h. Next, the supernatant was collected, and the protein concentration was determined using the BCA Protein Assay Kit. Equal amounts of protein samples were separated by 10% SDS-PAGE containing 0.1% gelatin. After electrophoresis, the gels were washed twice with 2.5% Triton X-100 to remove SDS, and the following processes were performed according to the protocol for the MMP Zymography Assay Kit (Applygen Technologies Inc., Beijing, China). Finally, the gel was stained with Coomassie Blue and scanned by a gel documentation system (Bio-Rad, CYMML-EPMY-028, American).
MDC staining
Autophagic vacuoles formed in the cells were detected by MDC staining. Cells (5 × 104) were plated onto cover slips in 24-well plates and incubated overnight to allow adherence. Then, the cells were treated with vehicle or with CAP (100 μM) and DDP (16.7 μM) alone or in combination for another 24 h. Then, the cells were washed three times with PBS and incubated with MDC (50 μM) for 30 min at 37 °C. Next, the excess MDC was removed, and the cells on the cover slips were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The autophagic vacuoles formed in the cells were analyzed by fluorescence microscopy with an excitation wavelength of 460–500 nm and an emission wavelength of 512–542 nm.
Transmission electron microscopy (TEM)
Autophagy induction by the different treatments was evaluated by examining autophagosome formation using TEM. MG63 cells were seeded into a 6-well plate at a density of 105 cells/wells overnight to allow adherence and then treated with CAP (100 μM) and DDP (16.7 μM) alone or in combination for another 24 h. Next, the cells were collected, fixed with 2.5% glutaraldehyde and 1% osmic acid, dehydrated with graded ethanol and acetone, embedded and sliced, and stained with 3% uranyl acetate-lead citrate. Finally, the cells were examined by TEM (JEM-1400 Plus, JEOL, Japan).
Measurement of intracellular ROS
Intracellular ROS was detected using 2,7-dichlorofluorescin diacetate (DCFH-DA). Briefly, cells (105 cells/well) were seeded into 6-well plates overnight to allow adherence and treated with various regimens for the indicated times. Then, the cells were washed with PBS three times and incubated in serum-free medium containing 10 μM DCFH-DA for 30 min at 37 °C in the dark. Next, the excess DCFH-DA was removed, and the cells were washed with PBS three times. The cells were observed immediately by fluorescence microscopy and then trypsinized, collected and detected by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.
Western blot
After cells were treated with different regimens for the indicated times, they were lysed in RIPA lysis buffer containing PMSF and phosphatase inhibitors to extract the total intracellular proteins. Protein samples (30–50 μg/lane) were separated on an 8–12% gel by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked with either 5% skim milk or 5% BSA at room temperature for 1 h and then incubated with the corresponding primary antibodies (1:800) overnight at 4 °C. After the membranes were washed with Tris-buffered saline with Tween-20 (TBST), they were incubated with a secondary antibody for 1 h at 37 °C. The membranes were washed, and the reactive protein bands were detected with an enhanced chemiluminescence (ECL) detection system and developed on film.
Xenograft tumor model
Twenty male nude mice (4 weeks old) were supplied by the Experimental Animal Center of Chongqing Medical University. All animal studies were approved by the Ethics Committee of Chongqing Medical University. The mice were housed with free access to a commercial diet and water under specific pathogen-free conditions. After the mice were acclimated for 1 week prior to study initiation, they were subcutaneously injected with 200 μL of sterile PBS containing a 143B cell suspension at a density of 106 cells/mL. After the tumor volume reached 50 mm3, treatment was initiated. Twenty mice were randomized into the following 4 groups (5/group): (1) CAP group, mice were administered CAP (20 mg/kg) in 200 μL of PBS via oral gavage; (2) DDP group, mice were administered DDP (4 mg/kg) in 200 μL of 0.9% saline solution via intraperitoneal injection; (3) CAP/DDP combinational group, both CAP and DDP were administered according to the aforementioned regimens, respectively; and (4) Control group, mice were left untreated. All groups received their respective treatments every 3 days for a total of seven treatments. The body weights of the mice were measured every 6 days. The tumor volume was measured every 3 days after treatment according to the following formula: 1/2 x a2b (a is the short axis and b is the long axis of the tumor). Mice were sacrificed under anesthesia on day 27, and the xenograft tumors from each animal were weighed and analyzed. To investigate the nephrotoxicity induced by the different regimens, blood samples in each group were collected before sacrifice to evaluate serum creatinine and blood urea nitrogen (BUN) levels. Mice kidneys were rapidly dissected out and fixed in 10% formalin for histopathological studies. Furthermore, to investigate the lung metastasis of the subcutaneous xenotransplanted tumor under the regimens, lungs from the rats were dissected out and fixed in 10% formalin for histopathological studies. After fixation, tissues from xenograft tumors, kidneys and lungs were dehydrated in a graded series of ethanol and xylene, embedded in paraffin, cut into sections, and stained with hematoxylin and eosin (H&E). The H&E-stained sections were examined under a light microscope at a magnification of 200X.
Immunohistochemistry (IHC)
IHC was performed to evaluate PCNA and Ki67 expression in xenograft tumor tissues. Briefly, the tumor tissues were separated, fixed with 4% paraformaldehyde, and embedded in paraffin. Next, the paraffin-embedded specimens were cut into serial sections (4 mm thick) by microtome. The tumor sections were blocked and immunostained with antibodies targeting Ki67 (1:200) and PCNA (1:200). Images were captured using a microscope, and PCNA and Ki67 expression were evaluated by counting the number of positive cells from 5 randomly selected fields in the residual viable tumor tissue among the necrotic areas under a light microscope at a magnification of 200X and 400X. Data are presented as the percentage of positive cells.
Discussion
DDP is used as the first-line chemotherapeutic agent in the treatment of OS, but its efficacy is limited by the development of resistance and normal tissue toxicity. Some conventional agents including methotrexate, doxorubicin and etoposide may be combined with DDP to improve efficacy and minimize toxicity, but multidrug resistance to these drugs should not be overlooked [
6]. Therefore, it is very important to seek a potential agent that can enhance the sensitivity of OS to conventional chemotherapeutic drugs. Due to their chemoprotective and anticancer characteristics, phytochemicals alone or together with other traditional chemotherapeutic agents have been introduced in the treatment of various cancers [
25,
26]. Previous studies have reported the use of phytochemicals with anticancer activities in combination with DDP to achieve greater efficacy for OS chemotherapy [
18,
27]. CAP is the most abundant and pungent component in a variety of hot peppers, and its anticancer effects have garnered increasing attention recently [
22]. Our previous study revealed that CAP inhibits the proliferation and colony formation of OS cells in a dose-dependent manner. However, apoptotic effects were only observed when the OS cells were exposed to relatively high concentrations of CAP (starting at 250 μM) [
23]. Furthermore, Jung et al. [
28] demonstrated that CAP has protective effects against cisplatin-induced renal dysfunction. These results suggest that CAP may exert therapeutic benefits as an adjunct to conventional chemotherapies but not as an independent anticancer agent. Therefore, we performed this study to assess the effects of using CAP combined with DDP on OS. In the present study, both CAP alone and DDP alone were validated to have inhibitory effects on OS cells in a dose-dependent manner. More importantly, our results further demonstrated that combination regimens with CAP and DDP at subtoxic concentrations showed significant synergistic anticancer effects (CI < 1) on OS cells. These findings suggested that CAP might be used as a chemotherapeutic agent to enhance anticancer effects in OS cells when combined with DDP.
Many chemotherapeutic agents primarily exhibit their anticancer effects by inducing apoptosis in cancer cells [
29]. However, the apoptosis induction efficacy of conventional agents gradually decreases due to chemoresistance [
30]. DDP resistance is mainly caused by inhibition of apoptosis of cancer cells [
31]. Therefore, an effective adjuvant to DDP-based chemotherapy should sensitize cancer cells to DDP-induced apoptosis. In the present study, subtoxic concentrations of CAP (100 μM) and DDP (16.7 μM) were selected to explore their combined effects on OS cells. The results showed that either CAP or DDP could induce indeterminate apoptosis in the three OS cells. However, the CAP/DDP combination induced apoptosis significantly. These results indicated that CAP is a potential candidate for strengthening the DDP-induced apoptotic effect in OS cells. The induction of apoptosis involves two classical pathways: the death receptor pathway (extrinsic pathway) and the mitochondrial pathway (intrinsic pathway) [
32]. The metabolic activities of mitochondria in cancer cells are higher than those in normal cells. Thus, the mitochondrial apoptotic pathway is considered an important target in cancer therapy [
33]. In the mitochondrial apoptotic pathway, loss of Δψm leads to an increase in mitochondrial membrane permeability, which leads to the release of cytochrome c from the mitochondria to the cytoplasm. In the cytoplasm, cytochrome c initiates the activation of caspases and eventually induces cell apoptosis. The release of cytochrome c is prevented by the Bcl-2 protein. In the present study, the Δψm in OS cells was decreased significantly in the CAP/DDP group compared with that in the control group. Furthermore, we observed a decrease in the antiapoptotic protein Bcl-2 with an increase in the proapoptotic protein Bax, and these changes were accompanied by an increase in cytochrome c in the CAP/DDP combination group. Furthermore, there were no significant changes when the control group was compared with either the CAP-alone or DDP-alone treatment groups. These results demonstrated that combination treatment using low concentrations of CAP and DDP could effectively induce apoptosis in OS cells through the mitochondrial apoptotic pathway.
Cell cycle progression is strictly supervised by a number of checkpoints that ensure the accuracy and integrity of DNA replication. When DNA is damaged by certain factors, these checkpoints initiate cell cycle arrest followed by activation of repair systems or induction of apoptosis. Evasion of cell cycle arrest is most frequently observed in tumor development [
34,
35]. One of the anticancer mechanisms of chemotherapeutic agents is the induction of cell cycle arrest in cancer cells [
36,
37]. It has been reported that the combination of other agents with DDP significantly induces various levels of cell cycle arrest in OS cells [
38,
39]. One of the major anticancer effects of CAP is to induce cell cycle arrest in OS cells [
23]. Therefore, we investigated the cell distribution in cell cycle phases after treatment with CAP and DDP alone or in combination. Our results revealed that CAP alone or in combination with DDP could induce G0/G1 cell cycle arrest in OS cells. However, the low concentration of DDP applied in the present study failed to inhibit cell cycle progression in OS cells. Moreover, the cell cycle-related proteins in OS cells also showed corresponding alterations. Although the combination of CAP and DDP inhibited the cell cycle transition by inducing G0/G1 arrest, the phenomenon and tendency were similar to those in the CAP-alone group. Our present results indicated that although synergistic suppression of cell viability was observed, the lower-concentration combination of CAP and DDP might not be able exert a synergistic effect on modulation of cell cycle progression.
Local invasion and distant metastases are frequently observed in OS patients, which is the major obstacle in the comprehensive treatment of OS [
40]. Emerging evidence suggests that MMPs play important roles in tumor invasion and metastasis and are abundantly expressed in various malignant tumors [
41]. Among the MMPs, MMP-2 and MMP-9 are essential for the initiation of migration and invasion [
42]. Previous studies have reported that CAP and DDP alone can suppress malignant cell invasion, which is associated with the downregulation of MMP-2 and MMP-9 [
38,
43,
44]. Thus, we performed Transwell migration assays of three OS cell lines to determine the effects of CAP and DDP as well as their combination on OS migration. Our results indicated that using CAP and DDP individually inhibited the invasion of OS cells, and the CAP/DDP combination treatment showed the greatest inhibitory effects. Furthermore, the inhibitory effects on migration and invasion were correlated with reduced expression and downregulation of the activation of MMP-2 and MMP-9. In conclusion, our results demonstrated that the use of CAP could significantly enhance the antimigratory effect of DDP on OS cells in vitro by suppressing the expression of MMP-2 and MMP-9.
Autophagy is an evolutionarily conserved process in which cytoplasmic materials and organelles are sequestered into autophagosomes and further degraded in the autophagolysosomes for recycling to maintain cellular homeostasis [
45]. Autophagy can be simulated by various factors, including starvation, growth factor deprivation, hypoxia and exposure to cytotoxic compounds. Substantial evidence has demonstrated the crucial role of autophagy in cancer initiation, differentiation, proliferation, metastasis and chemoresistance. Moreover, multiple studies have attempted to elucidate the paradoxical role of autophagy in cancer, which has provided a better understanding of autophagy in tumorigenesis and has established autophagy as a target for cancer treatment [
46]. A large number of phytochemicals and conventional chemotherapeutics have been shown to induce autophagy in various cancerous cells [
47,
48]. In the present study, we observed that the use of CAP or DDP alone could induce autophagy in OS cell lines via MDC staining and TEM. The LC3II/LC3I ratio, an indicator of autophagy, was significantly increased. Furthermore, we also observed an increase in the levels of various proteins involved in autophagosome formation, including Beclin1, Atg3, Atg5, and Atg16. Our present results revealed that using CAP or DDP alone could induce autophagy in OS cells and that the combination of CAP and DDP induced more significant autophagy than either agent alone. The AKT/mTOR signaling pathway plays crucial roles in many cellular functions such as cell growth, apoptosis and differentiation. Aberrant activation of the AKT/mTOR pathway is widely observed in various malignant tumors, which accelerates proliferation, increases resistance to apoptosis and promotes invasion and metastasis [
49]. Moreover, the AKT/mTOR signaling pathway has been reported to negatively regulate autophagy, and the inhibition of mTOR activation promotes autophagy [
50]. The present results showed that the expression of p-AKT and p-mTOR were significantly decreased after exposure to CAP alone, DDP alone or the CAP/DDP combined treatment, indicating suppression of the AKT/mTOR signaling pathway in OS cells. Furthermore, the inhibition was more significant with the combination of CAP and DDP than either CAP or DDP alone. In conclusion, our present results demonstrated that CAP and DDP or their combination induced autophagy in OS cells, and this process was negatively mediated by the ROS/AKT/mTOR signaling pathway.
The present findings are consistent with those of previous studies showing that many anticancer agents can induce autophagy in OS cells. However, the effects of autophagy in determining cancer fate, either by promoting cell survival or inducing cell death, are still controversial. Moreover, the precise crosstalk between autophagy and apoptosis has not yet been elucidated. To further investigate the promotional or antagonistic role of autophagy in mediating apoptosis in OS cells after treatment with CAP and DDP alone or in combination, we assessed cellular viability and apoptosis as well as the expression of related proteins after blocking autophagy with Baf-A1, a late-stage autophagy inhibitor that prevents the fusion of autophagosomes with lysosomes by inhibiting V-ATPase-dependent acidification [
46]. Our results showed that pretreatment with Baf-A1 decreased HOS cell viability and increased apoptosis compared with those in the groups that were not treated with Baf-A1. Western blots also showed that the use of Baf-A1 significantly increased the expression of proapoptotic proteins such as Bax, cytochrome c and cleaved caspase-3 and decreased the expression of the antiapoptotic protein Bcl-2 compared with treatment with CAP alone, DDP alone and the combination. Taken together, these findings strongly indicate that the induction of autophagy might play a prosurvival role in OS cells under the three different treatments in the current study.
ROS serve as an important molecular signal in various vital cellular processes, including proliferation, differentiation and apoptosis [
51]. Numerous lines of evidence have also demonstrated the critical role of ROS in the initiation and mediation of autophagy [
52]. Thus, we explored the role of ROS-mediated autophagy and apoptosis in HOS cells exposed to the CAP/DDP combination treatment. We observed a significant elevation of intracellular ROS after CAP/DDP combination treatment. However, in cells pretreated with NAC, a general ROS scavenger, the ROS elevation induced by CAP/DDP treatment was effectively reversed. Furthermore, the viability of HOS cells was preserved, and CAP/DDP-induced apoptosis was also partially suppressed. As the activation of ROS/JNK has been suggested to effectively induce apoptosis in cancerous cells [
53], to further explore the underlying mechanisms of ROS elevation in OS cells after treatment with the CAP/DDP combination, we assessed the key protein alterations involved in apoptosis, autophagy and the associated signaling pathways, including AKT/mTOR and ROS/JNK, with or without pretreatment with the ROS scavenger NAC. The present results showed that NAC modulated the ratio of Bax/Bcl-2 by downregulating Bax and upregulating Bcl-2, which resulted in decreased expression of cleaved caspase-3. These results were consistent with those obtained by flow cytometry, which showed that NAC inhibited the apoptosis induced by the CAP/DDP combination. In addition, NAC also decreased the expression of beclin1 and the conversion of LC3I to LC3II, which indicated that NAC could inhibit the autophagy induced by the CAP/DDP combination. All of these results demonstrated that the combination of CAP and DDP may trigger apoptosis and autophagy in OS cells by inducing ROS. Furthermore, our results also showed that ROS blockage by pretreatment with NAC could reverse the inactivation of AKT and mTOR in the AKT/mTOR pathway and inhibit JNK phosphorylation in the ROS/JNK signaling pathway. Taken together, all of the above results suggest that the combination of CAP and DDP triggers apoptosis and autophagy, which may be mediated by ROS through the inhibition of the ROS/Akt/mTOR pathway and activation of the ROS/JNK signaling pathway.
Finally, we further validated the effects of CAP and DDP in vivo by using xenograft models. Our results showed that tumor growth was effectively reduced in all treatment groups of 143B OS xenograft mice compared with that in the control group. In addition, the CAP/DDP combined treatment group showed the strongest inhibitory effects, consistent with the findings in vitro. Moreover, combined treatment with the two drugs did not significantly affect the body weight of the mice, and no significant nephrotoxicity were observed in any of the treatment groups of mice. These results indicated that this combination might be a relatively effective and safe regimen for OS.