Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in developing countries, especially in Eastern Asia [
]. Although the therapy strategies have been improved, ESCC is still one of the most aggressive types of cancer with poor prognosis and rapid progression and the 5-year overall survival rate less than 15%. Palliative chemotherapy with platinum-based regimen is generally indicated for patients with ESCC. Response rate to these platinum drugs is modest. Hence, there is a need for a novel treatment modality involving these anticancer drugs that selectively target cancer cells and circumvent treatment-resistant pathways for the management of ESCC.
Myeloid cell leukemia 1 (MCL-1) is a major prosurvival member of the Bcl-2 family proteins, which is mainly localized to the outer mitochondrial membrane via its C-terminal transmembrane (TM) domain [
]. Studies demonstrated the MCL-1 is important for cell proliferation, differentiation and tumorigenesis by regulating the apoptosis pathway [
]. By sequestering the proapoptotic multidomain proteins BAX and BAK, MCL-1 inhibits permeabilization of the mitochondrial membrane and ultimately preventing apoptosis. MCL-1 is subject to negative regulation by BH3-only protein family (e.g. NOXA, BIM and PUMA), which specifically bind to the BH3 binding groove, formed by BH domains of MCL-1, displacing MCL-1 from BAX and/or BAK and thus promoting apoptosis. MCL-1 frequently overexpressed in a variety of human tumor tissues, including stomach [
], liver [
], pancreas [
], prostate [
] and lung [
], which contributes to tumor development and progression and associated with poor patient prognosis. High expression of MCL-1 has been shown in human esophageal carcinoma cell lines including CE8 1 T/VGH, KYSE450, TE-1, Eca109, KYSE150 and KYSE510 [
]. However, whether MCL-1 is overexpressed in human primary ESCC tumors and contributes to ESCC development and progression remains unclear.
Cisplatin is frequently used for the treatment of various cancers, including ESCC, but some patients have a poor response to cisplatin-based chemotherapy. New strategies that could enhance chemosensitivity to cisplatin are needed. Overexpression of MCL-1 is frequently resistance to various cancer therapies, including chemotherapy [
]. Genetic silencing of
sensitizes a spectrum of cancers, such as melanoma, non-small cell lung and hepatocellular cancers to chemotherapy [
]. In addition, plenty of researches showed that the expression level of MCL-1 determines the sensitivity of multiple cancers to cisplatin. For instance, microRNA-193b enhances the cytotoxicity of cisplatin to hepatocellular carcinoma cells by targeting
]. Imperatorin acts as a cisplatin sensitizer via down-regulating MCL-1 expression in HCC chemotherapy [
]. The selective Wee-1 kinase inhibitor AZD-1775 sensitizes HPV-positive HNSCC cells to cisplatin-induced apoptosis in vitro accompanied by selective decrease in expression of MCL-1 and XIAP antiapoptotic proteins [
]. Knockdown of MCL-1 by siRNA or inhibition of MCL-1 by specific pharmacologic inhibitor EU-5148, sensitizes TWEAK-treated non-small cell lung cancer cells to cisplatin-mediated apoptosis [
]. Knockdown of MCL-1 also enhances sensitivity to cisplatin in gastric cancer cells expressing high levels of MCL-1 [
]. Considering high expression of MCL-1 in some ESCC cell lines [
], MCL-1 might function as an effective target to enhance the sensitivity of ESCC cells to cisplatin. However, whether MCL-1 inhibition acts as a cisplatin-chemosensitizing strategy in ESCC cells and the underlying mechanism remains incompletely defined.
In the current study, we found that MCL-1 expression was significantly increased in ESCC tissues compared to normal adjacent tissues and was associated with depth of invasion and lymph node metastasis. Moreover, MCL-1 inhibition by either genetical or pharmacological approach significantly enhanced the cytotoxicity of cisplatin to ESCC cells. The combination of UMI-77 and cisplatin induced apoptosis more significantly compared with treatment of UMI-77 or cisplatin alone by causing caspase-3 activation and PARP cleavage. In addition, the results demonstrated that UMI-77 prevented MCL-1/BAX and MCL-1/BAK complexes formation. To our knowledge, this is the first report to demonstrate that the chemosensitizing effect of a selective MCL-1 inhibitor UMI-77 combined with cisplatin to treat ESCC cells. The results suggested that MCL-1 is a promising therapeutic target for chemosensitization of ESCC cells to cisplatin and might provide a scientific basis for developing effective approaches to treatment human ESCCs.
Clinical tissue sample collections
Fresh tumor tissues and the corresponding normal adjacent tissues of the same patient with pathologically and clinically confirmed ESCC were collected from 49 patients by the Department of Cardiothoracic Surgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China. Several small pieces of fresh tumor tissue samples were dissected from the main tumor part of each surgically removed specimen. A portion of tumor and normal adjacent tissues were frozen immediately in liquid nitrogen and then stored at −80 °C for protein and mRNA extraction and analysis of MCL-1 expression by RT-PCR and Western blotting, respectively. A portion of tumor and normal adjacent tissues were fixed in formalin solution and sent for histological examination. The paraffin-embedded sections from the specimens, which were diagnosed as having ESCC, were used for immunostaining of MCL-1 protein expression. All tumors were confirmed as ESCC by the Clinicopathologic Department at the Second Xiangya Hospital of Central South University. All cases were classified according to the sixth edition of the pathologic tumor-node-metastasis (pTNM) classification. All the patients received no treatment before surgery.
Cell lines and culture
The KYSE150, KYSE510, Eca109, and TE-1 ESCC cell lines were obtained and grown in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics as previously reported [
]. Het-1A, a non-tumourigenic SV40T-immortalized human esophageal epithelial cell line [
], was purchased from the American Type Culture Collection (Manassas, VA). The 293 T cell line was obtained as previously reported [
]. Het-1A and 293 T cells were cultured with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 1% antibiotics. All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO
. Each vial of frozen cells was thawed and maintained for 2 months (10 passages). The cells were cultured for 36 to 48 h and proteins extracted for analysis.
Chemical reagents, including Tris, NaCl, and SDS, for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). UMI-77 (Cat. No. S7531, Selleck Chemicals) was dissolved in DMSO at 100 mM and stored in aliquot at −80 °C. Aliquots were diluted in corresponding medium just before addition to cell cultures. Cisplatin (Cat. No. 479306) was purchased from Sigma-Aldrich (St. Louis, MO). Stock cisplatin solution was prepared in DMSO at 200 mM stored as aliquots at −80 °C and used within 1 week and further diluted in medium before adding to the cells. The short hairpin RNAs (shRNAs) against human
Mcl-1 were purchased from Thermo Scientific. Two targeting sequences,
pLKO.1-shMCL-1#1, CCGGGCTAAACACTTGAAGACCATACTCGAGTATGGTCTTCAAGTGTTTAGCTTTTTG and
pLKO.1-shMCL-1#2, CCGGGCAGAAAGTATCACAGACGTTCTCGAGAACGTCTGTGATACTTTCTGCTTTTTG, were used in the study.
pLKO.1-shGFP (plasmid #30323), the lentiviral packaging plasmid
psPAX2 (plasmid #12260) and the envelope plasmid
pMD2.G (plasmid #12259) were available on Addgene (Cambridge, MA).
Cell proliferation assays
Cell proliferation assays were performed as previously described [
Anchorage-independent cell growth assays
Cells (8 × 10
per well) were seeded into 6-well plates with 0.3% Basal Medium Eagle agar containing 10% FBS and cultured. The cultures were maintained at 37 °C in a 5% CO
incubator for 2 or 3 weeks and colonies were counted under a microscope as previously described [
Protein preparation and Western blot analysis
Frozen tissue samples were sectioned into small pieces and dissolved in lysis buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1% NP-40. The samples were homogenized, sonicated and kept on ice for 30 min. After centrifugation, the supernatant was collected for immunoblotting analysis. Cultured cells were harvested and whole cell lysates were prepared according to the method previously described [
]. Protein concentration was determined using the BCA Assay Reagent (Cat. no. 23228, Pierce, Rockford, IL). Western blotting was performed as previously described [
]. Primary antibodies were used for immunoblotting: MCL-1 (#5453), cleaved caspase-3 (#9664), cleaved PARP (#5625), BCL-2 (#2870), BCL-xL (#2764), BAX (#5023) and BAK (#6947) from Cell Signaling Technology; β-actin (A5316) from Sigma-Aldrich; GAPDH (sc-47,724) from Santa Cruz Biotechnology. Secondary antibodies were anti-rabbit IgG HRP (#7074) and anti-mouse IgG HRP (#7076) and purchased from Cell Signaling Technology. Antibody conjugates were visualized by chemiluminescence (ECL; cat#34076, Thermo).
mRNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from frozen specimens using Trizol reagent (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized from 2 μg of total RNA using the Reverse Transcription System Kit (Cat. No. A3500, Promega, Madison, WI). The resulted cDNA was subjected to PCR (95 °C for 5 min followed by 36 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 40 s, and an extension for 10 min at 72 °C) using primers designed for human
]: sense, 5′-cggcagtcgctggagattat-3′ and antisense, 5′-gtggtggtggttggtta-3′, yield a 573-bp product; or for
: sense, 5′-ttccagccttccttcctggg-3′ and antisense, 5′-ttgcgctcaggaggagcaat-3′, yield a 224-bp product. PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide.
Immunohistochemical (IHC) staining
Tumor tissues obtained from ESCC patients or euthanized xenografted mice were embedded in paraffin and subjected to immunohistochemistry staining with specific antibodies against MCL-1 (1:100, sc-819, Santa Cruz Biotechnology) or Ki67 (1:200, ab16667, Abcam) according to the DAKO system protocol. Hematoxylin was used for counterstaining. Slides were viewed and photographed under a light microscope, and analyzed using Image-Pro Plus Software (version 6.2) program (Media Cybernetics).
The generation of gene stable knockdown cell lines was performed as described previously [
]. Briefly, to generate MCL-1 knocking down cells,
lentivirus plasmids were cotransfected into 293 T cells with
. Viral supernatant fractions were collected at 48 h after transfection and filtered through a 0.45 μm filter followed by infection into KYSE150 and KYSE510 cells together with 6 μg/mL polybrene. At 16 h after infection, the medium was replaced with fresh medium containing 2 μg/mL puromycin and cells were incubated for another 6 days. For transient knockdown MCL-1, KYSE150 and KYSE510 cells were grown in 6-well plates and transfected with an
siRNA (Cat. No. SC-35877; Santa Cruz Biotechnology) or a control siRNA (Cat no. sc-37,007; Santa Cruz Biotechnology) using HiPerFect transfection reagent (Cat no. 301705, Qiagen) for 48 h according to the manufacturer’s instructions. Cells were then harvested for protein extraction and immunoblotting to confirm MCL-1 knockdown.
Co-immunoprecipitation (co-IP) assays
Co-IP assays were performed as described previously [
]. Briefly, cells were serum-starved in 0.1% FBS/RPMI 1640 medium overnight followed by treatment with DMSO or the indicated concentrations of UMI-77 for 48 h. Cells were harvested in IP lysis buffer (Cat. No. 87788, Thermo Scientific) as described by the manufacturer. Cell lysates were pre-cleared with 40 μl of protein A/G-agarose beads (sc-2003, Santa Cruz Biotechnology) and immunoprecipitated with 2 μg of anti-MCL-1 (sc-819, Santa Cruz Biotechnology) or normal rabbit IgG (NI01, Calbiochem) at 4 °C overnight, followed by 2 h of incubation at 4 °C with 40 μl of protein A/G-agarose beads. Immunocomplexes were resolved by SDS-PAGE and co-immunoprecipitated proteins were detected using anti-BAX (#5023, Cell Signaling Technology) and anti-BAK (#6947, Cell Signaling Technology) antibodies, respectively.
In vivo tumor growth assay
All mouse studies were performed utilizing protocols approved by the Institutional Animal Care and Use Committee of the Second Xiangya Hospital of Central South University. Athymic nude mice (BALB/c nude mouse, 6 wk. old) were randomly divided into two groups (
= 10) and subcutaneously injected in the flank with KYSE150-shGFP or KYSE150-shMCL-1#2 esophageal carcinoma cells (2 × 10
). Mice were weighed and tumors measured by caliper every other day. Tumor volume was calculated from measurements of 2 diameters of the individual tumor according to the following formula: tumor volume (mm
) = (length × width × width/2) [
]. Mice were monitored until day 27 and at that time mice were euthanized and tumors extracted.
Statistical analysis was done with the statistical software program SPSS ver.12.0. The statistical significance of the correlations between MCL-1 overexpression and clinicopathologic characteristics were assessed by
2 test or Fisher’s exact test. Results expressed as mean ± SD were analyzed using the Student’s
t test. Differences were considered significant when
p < 0.05.
Expression of antiapoptotic protein MCL-1 is frequently elevated in various human tumors. MCL-1 thus appears to be an attractive direct target for anticancer therapy. Pharmacological agents that cause MCL-1 depletion have been extensively investigated for anticancer therapy. Several pharmacological agents have been shown to diminish MCL-1 expression by inhibiting MCL-1 production or enhancing MCL-1 degradation. For instance, various CDK inhibitors, such as flavopiridol [
], roscovitine [
and SNS-032 [
], diminish MCL-1 levels and induce apoptosis in a variety of cell types. AZD8055, an mTORC1/2 inhibitor, reduces MCL-1 expression in
-mutant colorectal cancer cells [
]. The USP9X inhibitor WP1130 lowers MCL-1 levels in chronic myelogenous leukemia and enhances sensitivity to apoptosis by facilitating MCL-1 degradation [
]. In addition, BH3 mimetics that block the hydrophobic BH3-binding groove of MCL-1 have been developed, which mimic the BH3 domain and therefore are able to fit into the hydrophobic pocket of MCL-1 and block its ability to bind proapoptotic proteins, inhibiting their function. These small molecules include gossypol [
], obatoclax (GX15–070) [
], sabutoclax (BI97C1) [
], and BH3-M6 [
]. However, the above-mentioned small molecule BH3 mimetics are lack of selectivity for MCL-1 [
], which bind to not only MCL-1, but also other Bcl-2 family proteins, BCL-xL or/and BCL-2. Major efforts have been invested and progress has been made in developing specific inhibitors of MCL-1. Compound A-1210477, a derivative of indole-2-carboxylic acids, has been found to selectively and directly bind MCL-1, induce intrinsic apoptosis and demonstrate single agent killing of multiple myeloma and NSCLC cell lines [
]. The MCL-1-specific inhibitor UMI-77, which selectively binds to the BH3-binding groove of MCL-1, inhibits cell growth, induces apoptosis in pancreatic cancer cells and effectively inhibits BxPC-3 xenograft tumor growth [
]. Our results indicated that UMI-77 also induced apoptosis in ESCC cells when administrated as single agent
by the disruption of MCL-1 binding to BAX and BAK
. Therefore, targeting MCL-1 might qualify as a promising novel approach in ESCC therapy.
Clinically, chemotherapy is one of the most important therapeutic methods to treat numerous cancers. Cytoxic agents such as platinum (e.g. cisplatin), fluorinated pyrimidines (e.g. 5-Fu) and taxanes (e.g. paclitaxel) drugs are widely administered for chemotherapy to treat various types of cancer including ESCC, but poor response to cytoxic agent-based chemotherapy is not uncommon [
]. In view of the roles of MCL-1 in tumorigenesis, tumor progress and chemoresistance, the combinations of MCL-1 inhibitors with classical cytoxic agents have been actively investigated. It has been reported that obatoclax (GX15–070) induces apoptosis and enhances cisplatin-induced apoptosis in NSCLC cells [
]. Ren et al. [
] reported that (−)-Gossypol enhances the antitumor efficacy of cisplatin through inhibition of APE1 repair and redox activity in non-small cell lung cancer. Furthermore, synergistic antitumor effects have been observed when MCL-1 inhibitors combined not only with cytoxic drugs but also with other chemotherapeutic agents. For instance, MCL-1 inhibitor sabutoclax (BI97C1) and COX-2 inhibitor celecoxib synergistically inhibits the growth of oral squamous cell carcinomas cells both in vitro and in vivo [
]. Sabutoclax also reportedly synergizes with minocycline to induce growth arrest and apoptosis in pancreatic cancer cells [
]. Our results demonstrated that UMI-77 synergistically enhanced cisplatin-induced apoptosis in both KYSE150 and KYSE510 cells
. Since MCL-1 was overexpressed in more than 60% of ESCC patient samples
, it might contribute to poor response to chemotherapy in some of the ESCC patients. The enhanced apoptosis when cisplatin in combination with MCL-1 knockdown
or with MCL-1 inhibitor UMI-77
further suggested that the combination of cisplatin with other therapies that modulate MCL-1 could be exploited as a plausible strategy to enhance therapeutic efficacy for ESCCs.
Our results indicated that, among the ESCC cell lines evaluated, KYSE510 cell line with the highest level of MCL-1 and the lowest level of BCL-xL exhibited high susceptibility to UMI-77-induced apopotosis. However, KYSE150 cell line expressing similar level of MCL-1 and higher level of BCL-xL displayed less sensitivity to UMI-77 treatment compared with KYSE510 cell line
1a and b
. As the survival of most tumors is not dependent on a single antiapoptotic Bcl-2 protein, strategies that combination of MCL-1 inhibition with inhibitors targeting different Bcl-2 family members would be more successful than therapies targeting only a single antiapoptotic Bcl-2 family protein. For instance, down-regulation of MCL-1 enhances cell-killing abilities of ABT-737 and ABT-263, which both inhibit BCL-2 and BCL-xL but not MCL-1 [
]. MCL-1 down-regulation by CDK inhibitor roscovitine or
-shRNA dramatically increases ABT-737 lethality in human leukemia cells [
]. Faber et al. [
] reported that the combination of ABT-263 and AZD8055, an mTORC1/2 inhibitor that reduced MCL-1 protein levels, potently suppresses tumor progression across a variety of preclinical small cell lung cancer experimental models. Potent and selective small-molecule MCL-1 inhibitors A-1210477 synergizes with the BCL-2 and BCL-xL inhibitor ABT-263 to kill a variety of cancer cell lines [
]. In some cancer types, especially for those cell lines that rely on multiple Bcl-2 family members for survival, efficient treatment will more commonly require either a pan-Bcl-2 family protein inhibitor or a combination of inhibitors that neutralises the different Bcl-2 family members, which could be a rational approach in treating tumors.
Mechanistically, apoptosis induction by UMI-77 is BAX/BAK-dependent, preceded by disrupting disruption of MCL-1 binding to BAX and BAK
. Although we did not investigate the activity of UMI-77 in animal tumor models, previous study by Abulwerdi et al. [
] have shown that UMI-77 is well-tolerated and inhibits the growth of pancreatic tumor xenografts with no apparent toxicity in normal mouse tissues. This study revealed the presence of TUNEL-positive apoptotic cells in tumors collected from UMI-77-treated animals, further supporting our observations that induction of apoptosis is, at least in part, a mechanism of action of UMI-77. Since the in vitro data in our present study demonstrated the efficacy of UMI-77 as a chemosensitizing agent in ESCC cells, it will be important in future studies to determine the anti-tumor effects as well as toxicity of the combination of UMI-77 with cytotoxic drugs or other chemotherapeutic agents in animal models.
It has been well examined that the multidomain proapoptotic proteins BAK and BAX are executors of the mitochondrial pathway of apoptosis whose activation can be prevented by antiapoptotic Bcl-2 family proteins such as MCL-1 and BCL-xL [
]. Although BAX and BAK seem in most circumstances to be functionally equivalent, substantial differences exist. BAX is largely cytosolic, whereas BAK resides in complexes on the outer membrane of mitochondria and on the endoplasmic reticulum of healthy cells. Nevertheless, the activation of BAX and BAK appears similar [
]. On receipt of cytotoxic signals, both BAX and BAK change conformation, and BAX translocates to the mitochondrial outer membrane, where both BAX and BAK then form homo-oligomers that can associate, leading to membrane permeabilization [
]. In some cell types, such as chronic myelogenous leukemia [
] and multiple myeloma [
], the forced reduction of MCL-1 permits BAK oligomerization, activation and is sufficient enough to induce apoptotic cell death. However, some types of cells require a second signal such as genotoxic stress to induce apoptosis [
]. This difference may be accounted for the different level of BCL-xL, because BCL-xL may replace MCL-1 in its suppression of BAK activation [
]. In the case of BAX, it does not co-immunoprecipitate with MCL-1 or BCL-xL in HeLa cells [
]. However, the apoptosis induced by MCL-1 suppression was partially mediated through BAX in rheumatoid arthritis synovial fibroblasts [
] and in pancreatic cancer cells [
]. Our results indicated that KYSE510 cells expressed lower BCL-xL protein level than KYSE150 cells
1a and b
which accompanied by a stronger response to cisplatin- or UMI-77-induced apopotosis than did KYSE150 cells
. The detail mechanism by which BCL-xL replaces MCL-1 and suppresses BAK activation and whether coordinately targeting both MCL-1/BAK axis and BCL-xL/BAK axis heighten the sensitivity of ESCC cells to cisplatin-induced apoptosis need to be further investigated.