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Colorectal cancer is the most common malignancy of the gastrointestinal tract. Surgical treatment combined with radiotherapy is the main treatment course for colorectal cancer; nevertheless, radio-resistance is commonly encountered during the treatment course and seriously influences the therapeutic efficacy. We tested the hypothesis that the CXCL12/CXCR4 axis is closely related to radiotherapy sensitivity in colorectal cancer cells. Here, we found that the decrease in cell viability and the increase in cell death induced by radiotherapy were attenuated by CXCL12 treatment, and the inhibition of CXCR4 promoted colorectal cancer cells to be more sensitive to radiotherapy. We also examined the critical roles of CXCL12/CXCR4 in cell survival and found that radiotherapy induced Bax expression and facilitated the activity of caspase-3 and caspase-9, which were reversed by CXCL12 treatment. Cell apoptosis was enhanced by the inhibition of CXCR4 under radiotherapy conditions. Furthermore, treatment with CXCL12 resulted in an increased expression of survivin, and the inhibitory roles of CXCL12 in radiotherapy-induced apoptosis were mitigated by survivin knockdown. These results indicate that CXCL12/CXCR4 protects colorectal cancer cells against radiotherapy via survivin, implying an important underlying mechanism of resistance to radiotherapy during colorectal cancer therapy.
Colorectal cancer (CRC), whose 5-year survival rate is less than 30%, is the most common malignancy of the gastrointestinal tract.1 Currently, the principle and standard treatment for advanced colorectal cancer is surgical resection combined with radiotherapy (RT) and chemotherapeutic therapy.2,3 However, the frequent existence of resistance to radiotherapy in colorectal cancer cells during radiotherapy, which significantly affects and impairs the therapeutic effect, causes cancer cells to avoid death. Thus, it is necessary to study the critical factors involved in regulating the sensitivity of colorectal cancer cells to radiotherapy to improve the clinical therapeutic outcomes.
Stromal cell-derived factor 1 (CXCL12, also known as SDF-1) is an inducible chemokine that belongs to the human chemokine superfamily.4 CXCR4 is the most common chemokine receptor of CXCL12 in various types of cells, and the biological roles of CXCL12 in cell growth, differentiation, and angiogenesis are accomplished via binding to its receptor (CXCR4).5,6 It has been reported that elevated expression of CXCR4 is correlated with a significant reduction in median overall survival in renal and cervical cancer.7,8 The expression of CXCR4 is also frequently upregulated in the primary tumors of CRC patients.9,10 Moreover, recent studies have indicated that the CXCL12-CXCR4 pathway is closely related to the initiation and progression of some tumors, and inhibiting the CXCL12-CXCR4 pathway is being considered as a way to sensitize tumors towards anticancer therapies.11–13 However, the roles of CXCL12/CXCR4 in the radiotherapy sensitivity of colorectal cancer cells and the corresponding molecular mechanisms remain unclear.
Apoptosis is a major pathway of cell death, and an increase in cell survival largely results from the inhibition of cellular apoptosis. The overexpression of anti-apoptotic molecules in cancer cells is an important mechanism underlying tumor radio-resistance and recurrence. Survivin, belonging to the inhibitor of apoptosis (IAP) family, is a widely expressed anti-apoptotic protein; it participates in regulating the development and progression of many tumors because of its capacity to facilitate cell survival, promote cell cycle progression, and enhance aggressive tumor behavior.14,15 Previous studies have indicated that the increased level of survivin is closely related with cancer progression and radio-resistance in some cancers, and the inhibition of survivin is considered to be a candidate target for cancer molecular therapeutics.16,17 However, it is still necessary to explore the precise role of survivin in radiotherapy treatment and the regulatory mechanisms of survivin in colorectal cancer cells.
The purpose of our research is to determine whether the CXCL12/CXCR4 axis is closely associated with the sensitivity of radiotherapy in colorectal cancer cells and elucidate the corresponding molecular mechanisms. In this study, our results have shown that radiotherapy-inhibited cell growth is mitigated by CXCL12 treatment, whereas the inhibition of CXCR4 promotes colorectal cancer cell sensitivity to radiotherapy. Moreover, the activation of the CXCL12/CXCR4 pathway leads to the radio-resistance of colorectal cancers cells by upregulating the expression of survivin. These findings provide a novel strategy and potential target for improving the therapeutic efficiency of radiotherapy in colorectal cancer.
The Bax polyclonal antibody (1:500), the Survivin monoclonal antibody (1:500), and the beta-actin monoclonal antibody (1:5000) were all obtained from Santa Cruz Biotechnology. Caspase-3 and caspase-9 activity assay kits and the cytotoxicity assay kit were purchased from the Beyotime Institute of Biotechnology. CXCL12 was got from Peprotech.
HCT116 cells were acquired from ATCC (Rockville, Maryland, USA). We routinely cultured the cells at 37 in DMEM medium supplemented with 10% fetal bovine serum in a humidified atmosphere with 5% CO2.
IR treatment was conducted using an irradiator with a cobalt Co60 source (Co-V, Theratron 780; MDS Nordion, Ottawa, Ontario, Canada) at a dose rate of 1 Gy/min.
Survivin small interfering RNAs (siSurvivin) and non-targeted control siRNA (sicontrol) were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). HCT116 cells were transfected with siSurvivin or sicontrol. Briefly, we seeded HCT116 cells on a plate and cultured them until 30–50% conﬂuency. Then, we diluted 2.0µg of siRNAs and 10.0µL of X-treme with serum-free Opti-MEM-1 media. After softly mixing them, the mixture was allowed to stand for 20min at 25. After that, the mixture was added to the plates.
The assessment of cell viability was determined by the CCK-8 reagent (Dojindo, Japan). HCT116 cells were digested by trypsin and seeded onto a 96-well plate. The density of each well was 2×103 cells. After starvation for 24h, 100µL of culture media with the indicated treatments were added into cells. After 48h incubation, we then added 10µL of the CCK-8 reagent into each well and kept them at 37 for 2h. After that, a microplate reader at 450nm was used to measure the absorbance.
A cytotoxicity detection kit was utilized to detect the release of LDH. Briefly, after the indicated treatment, we added 120µL of supernatant into a new 96-well plate and added 60µL of LDH detection solution from the kit into each well. After 30min of incubation at 25 in the dark, a microplate reader at 490nm was used to measure the absorbance.
A caspase-3 activity assay kit was utilized to detect its activity. Briefly, cells were solubilized with 100µL of lysis buffer from the kit for 15min on ice. Then, 50µg of protein from the lysates was moved into a new tube, and the reaction buffer with Ac-DEVD-pNA, prepared in accordance with the manufacturer instructions, was added to the tube. After incubation for 4h at 37, its absorbance at 405nm was measured by a microplate reader.
The procedure and steps were similar to the measurement of caspase-3 activity, but the chromogenic caspase substrate of caspase-9, Ac-LEHD-pNA, was used.
After washing with PBS, 1×106 cells were treated with RIPA buffer for 20min on ice. The lysates were centrifuged at 12,000g for 10min at 4. Protein concentration was evaluated by the Bradford protein assay. A total of 50µg of protein from each group was then loaded into 12% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred from the gels to a PVDF membrane. The membrane was then incubated in 3% BSA for 1h and then allowed to react with the primary antibodies overnight at 4, followed by reaction with the secondary antibody for 1 hour at 25 after washing with PBS containing 0.1% TWEEN-20. The membrane was then treated with enhanced chemiluminescence reagents.
Data were represented as the mean values±SEM, and Student's t-test or one-way ANOVA followed by Dunnett’s test, where appropriate, was utilized for evaluating statistical significance. P<0.05 was considered to be statistically significant.
The frequent existence of radio-resistance significantly impairs the therapeutic effects of cancers. To determine whether the CXCL12/CXCR4 pathway is associated with the resistance to radiotherapy in colorectal cancers cells, we first treated HCT116 with exogenous CXCL12 and exposed them to radiotherapy. The CCK-8 results showed that radiotherapy reduced cell viability in a dose-dependent manner, and 8Gy radiotherapy obviously decreased cell viability in the HCT116 cells. However, the decrease in cell viability by radiotherapy was reversed by CXCL12 treatment (Figure 1(a)). An LDH assay was used to detect cell death, and we found that the release of LDH was potentiated by 8Gy radiotherapy, whereas CXCL12 treatment mitigated the effects of radiotherapy on LDH release (Figure 1(b)). These results indicate that CXCL12 treatment increases colorectal cancer cell survival after radiotherapy.
CXCR4 is the most important receptor of CXCL12. Subsequently, we utilized an inhibitor of CXCR4 (AMD3100) to examine the roles of CXCR4 during radiotherapy. We found that HCT116 cell viability was reduced by a minimum dose of 8Gy of radiation, with a concomitant increase in LDH release. However, after inhibiting CXCR4 with AMD3100, the decrease in cell survival and the increase in cell death were elicited at a lower dose of 4Gy of radiation, and even more cells were killed by the radiotherapy compared to the groups without treatment with AMD3100 (Figure 2(a) and 2(b)). These results indicate that the inhibition of CXCR4 promotes colorectal cancer cell sensitivity to radiotherapy.
It is well known that the suppression of tumor progression and the death of cancer cells caused by radiotherapy largely result from an increase in cellular apoptosis; we thus examined whether the CXCL12/CXCR4 pathway participated in regulating cell apoptosis from radiotherapy. Our results showed that 8Gy radiotherapy led to increased caspase-3 activity, whereas treatment with exogenous CXCL12 repressed the activation of caspase-3 induced by radiotherapy (Figure 3(a)). Moreover, radiotherapy led to an increase in caspase-9 activity and induced the expression of Bax, which was attenuated by CXCL12 treatment (Figure 3(b) and ((c)).c)). These results indicate that radiotherapy-facilitated cellular apoptosis is antagonized by CXCL12 treatment in colorectal cancer cells.
We utilized AMD3100 to block the physiological functions of the CXCL12/CXCR4 pathway. We observed that the apoptotic changes caused by radiotherapy, including the activation of caspase-3 and caspase-9 and the increased protein expression of Bax, were enhanced by the inhibitor of CXCR4, indicating that radiotherapy-induced cell apoptosis and death were strengthened after the inhibition of the CXCL12/CXCR4 pathway (Figure 4(a) to ((c)).c)). These results imply that suppressing the CXCL12/CXCR4 pathway renders colorectal cancer cells more sensitive to radiotherapy.
Survivin, which is overexpressed in most cancer tissues, regulates the onset and development of cancer via acting as an inhibitor of apoptosis, and caspase-mediated apoptosis is significantly inhibited by the increased expression of survivin. Given our data that CXCL12/CXCR4 renders colorectal cancers cells resistant to radiotherapy by antagonizing cell apoptosis, we examined whether there is a linkage between CXCL12/CXCR4 and survivin. Our results showed that the expression of survivin was upregulated by treatment with exogenous CXCL12, whereas the inhibition of CXCR4 repressed the expression of survivin in HCT116 cells (Figure 5(a) and 5(b)). Survivin siRNA was utilized to knockdown the expression of survivin (Figure 5(c)). We found that the protective roles of CXCL12 in cell survival were attenuated by survivin siRNA under conditions of radiotherapy treatment (Figure 5(d)). Similar results were acquired when we measured cell apoptosis. We also found that the CXCL12-mediated attenuation of caspase-3 and caspase-9 activity induced by radiotherapy was mitigated by the knockdown of survivin (Figure 5(e) and ((f)).f)). The results indicate that the radio-resistance triggered by CXCL12 treatment is mediated by survivin in colorectal cancer cells.
Colorectal cancer is the most common malignant tumor of the gastrointestinal tract. Surgical treatment combined with radiotherapy is one of the most important modalities for the treatment of tumors, but radio-resistance, which commonly occurs during the course of treatment, seriously affects the therapeutic efficacy of the treatment and the prognosis of the patients with colorectal cancer. Unfortunately, the mechanisms behind radio-resistance in colorectal cancer are still largely unknown. Thus, it is necessary to explore the critical factors and related signaling pathways that regulate the sensitivity to radiotherapy in colorectal cancer cells. The present study demonstrates that the activation of the CXCL12/CXCR4 pathway renders colorectal cancers cell less sensitive to radiotherapy via up-regulating survivin expression.
It has been reported that the interaction of CXCL12 with CXCR4 leads to crucial signaling events not only in normal tissue but also in cancer cells.18,19 In colorectal cancers, the expression of CXCR4 is upregulated in the cancerous tissues. Moreover, the high expression of CXCR4 is correlated with a significant reduction in median overall survival.20–22 Accumulating evidence has indicated that the CXCL12/CXCR4 pathway participates in regulating the development of colorectal cancer. Nevertheless, the effects of CXCL12 and CXCR4 on cellular survival in colorectal cancer cells exposed to radiation are uncertain. In our study, exogenous CXCL12 attenuates the decrease in cell viability and the increase in cell death caused by radiotherapy. In contrast, more cells were killed by radiotherapy after inhibiting the CXCL12/CXCR4 pathway with AMD3100 (a competitive inhibitor of CXCR4). These results indicate that activation of the CXCL12/CXCR4 axis renders colorectal cancers cell less sensitive to radiotherapy, and inhibition of the CXCL12/CXCR4 pathway can promote colorectal cancer cell sensitivity to radiotherapy and augment the therapeutic effects of radiotherapy in colorectal cancer.
Apoptosis plays a key role in regulating cell growth, and radiotherapy induces cell death and inhibits cell growth partly through promoting cellular apoptosis.23 To emphasize the essential role of the CXCL12/CXCR4 axis in the radio-resistance of colorectal cancer cells, we examined whether the pathway regulated radiotherapy-induced apoptosis. Mitochondria-dependent apoptosis is an important subtype of cellular apoptosis, which is initiated by a decrease in the mitochondria membrane potential. Bax, localized on the outer membrane of mitochondria, participates in regulating the change of mitochondria membrane potential.24 The increased expression of Bax is associated with a decrease in mitochondria membrane potential, and caspase-9 is its downstream effecter molecule. The activation of caspase-9 subsequently increases the activity of caspase-3 (the executor of cell apoptosis).25–27 In the present study, our results showed that radiotherapy facilitated the expression of Bax and led to the increased activity of caspase-9 and caspase-3, whereas these pro-apoptotic changes induced by radiotherapy were attenuated by treatment with exogenous CXCL12. Moreover, CXCR4 inhibition enhanced cellular apoptosis induced by radiation exposure. These results show that the activation of the CXCL12/CXCR4 pathway inhibits cell apoptosis and promotes cell survival during radiotherapy.
Survivin, an important anti-apoptotic protein, belongs to the inhibitors of apoptosis protein (IAP) family.28 Previous studies have shown that the increased expression of survivin can significantly protect against p53 and caspase-mediated apoptosis.29,30 In consideration of the inhibitory effects of CXCL12/CXCR4 on radiotherapy-induced cellular apoptosis, we determined whether there is a linkage between CXCL12/CXCR4 and survivin. We found that the activation of the CXCL12/CXCR4 pathway resulted in increased levels of survivin, and the inhibition of this pathway had the opposite effects. Moreover, the knockdown of survivin mitigated the inhibitory roles of CXCL12 in cellular apoptosis. All of these results imply that the radio-resistance in colorectal cancer cells triggered by the activation of the CXCL12/CXCR4 pathway is, at least in part, mediated by survivin.
In conclusion, this study indicates that activation of the CXCL12/CXCR4 pathway inhibits cellular apoptosis and promotes cell survival via upregulating the expression of survivin in cells exposed to radiation and that blocking the CXCL12/CXCR4 pathway makes colorectal cancer cells more sensitive to radiotherapy. These findings suggest an important underlying mechanism for modulating the sensitivity to radiotherapy in colorectal cancer cells, which may provide a novel strategy and potential target for improving the therapeutic efficiency of radiotherapy in colorectal cancer.
DW, CJ and ZC conceived and designed the experiments; DW, YZ, DL, MZ, XM, JG, YH, WL and JH performed the experiments and analyzed the results; CJ, ZZ and ZC prepared the submission together.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
This work was supported by grants from the China Postdoctoral Science Foundation, China (grant no. 2012M520769 to Dawei Wang), the Hei Long Jiang Postdoctoral Foundation, China (grant no. LBH-Z12201 to Dawei Wang), the First Affiliated Hospital of Harbin Medical University Research Foundation (grant no. 2013B02 to Dawei Wang), the Heilongjiang Province Department of Education Science and Technology Research fund project (grant no. 12541814 to Zhuoxin Cheng), the Natural Science Foundation of Heilongjiang Province (grant no. H201373 to Zhuoxin Cheng), and the Natural Science Foundation of Heilongjiang Province (grant no. QC2013C101 to Jie Hou).