| BackgroundColorectal cancer is a common malignant tumor of the digestive system, which is the third common gastrointestinal tumors, and with the changes of people’s diet structure the incidence of colorectal cancer is increasing gradually. In recent years, the majority of patients have been detected early and treated appropriately, however, even resection of the primary tumor is in the early course of the disease, approximately 40% of patients suffered from peritoneal recurrence and liver metastasis within five years. The 5-year survival rate of patients suffered from colorectal cancer with liver metastasis is only 16.8% even with liver removing surgery at the same line,which are great harm to people’s health. Surgery is still the most effective way to treat colon cancer, but the symptoms of most colonrectal cancer are not typical and occult, which causes many patients missing surgery opportunity. Considering to reduce serious adverse reaction of chemotherapy, radiation therapy and traditional Chinese medicine have gradually become the research focus in the treatment of locally advanced colorectal cancer.The combination of Chinese and western medicine treatment of the colorectal cancer and other malignancies is one of the important measures to comprehensive treatment of tumors which has been gradually accepted and attract more and more attention. Epieriocalyxin A (EpiA) and eriocalyxin B (EriB) are two Kaurane diterpenoids collected from plant extract of Isodon eriocalyx planted in jiangchuan county of yunnan province. For a long time eriocalyxin B has been widely studied in clinical anti-cancer field, however, the anti-cancer research about epieriocalyxin A were few, and whether diterpenoids Epieriocalyxin A could be a potential anti-cancer agent and the molecular mechanisms remains unknown. Chinese anti-tumor medicine targeting the signal transduction pathway molecules have become a hot topic for the superiority of low toxicity, high efficacy and few chemoresistance. Our previous preliminary experiments found EpiA could induce apoptosis in colonrectal cancer Caco-2 cells, and as an extracellular stimulating factor by exactly which signal transduction pathway does EpiA start the colonrectal cancer cells apoptosis pathway is not yet clear. In-depth study of EpiA and revealing the specific signal transduction pathways help determine the role of EpiA upon intracellular targets and its antitumor molecular mechanism.Foreign large meta-analysis showed preoperative adjuvant radiation therapy has significant curative effect on rectal cancer with local recurrence. The volume of tumor reduced obviously and radical resection became reality after preoperative radiotherapy; The opportunities of postoperative distant metastasis and local recurrence are decreasing; The number of metastasis lymph node around the tumor are decreasing significant; Preoperative radiotherapy has an obvious degradation effect on patients suffered from rectal cancer. But quite a considerable amount of patients is not sensitive to radiatherapy and the followed adhesion after radiotherapy restrict the development of preoperative radiotherapy. The inherent and iatrogenic biology nature of malignant colorectal cancer has caused radiotherapy resistance becoming a formidable obstacle in clinical treatment, and only a small percentage of patients really have benefitted from radiotherapy caused by radiotherapy resistance.Adjuvant chemotherapy and targeted therapy are often used to improve the radiotherapy sensitivity of radiotherapy during comprehensive clinical treatment. Exploring the solution to increasing the radiotherapy sensitization of patients suffering from colorectal cancer presents a huge challenge to clinical and basic research.The occurrence of radiotherapy resistance is a complex process, involving excessive expression of DNA repair protein, multiple abnormal activation of signaling pathways, angiogenesis, tumor stem cells and autophagy. Christine Wild- Bode, etc. have found that the lethal dose of radiation could enhance the invasion and migration of human malignant glioma cell, and this malignant biological behavior via P53 non-dependent pathway, significantly increasing the expression of integrin αvβ3, obviously increased MMP-2 and MMP-9 expression levels and activity, reduced MT1-MMP and MMP-2 inhibitors (tissue inhibitor of matrix metalloproteinase-2) TIMP-1 expression and activity by upregulating Bcl-2/Bax ratio of inhibition apoptosis in human glioblastoma causing radiation resistance. Our Task Force confirmed upregulation of integrin αvβ6 could protect colon cancer cells from apoptosis and promoting the invasion and migration, but the mechanism of integrin avP6 involved in the radiotherapy resistance of colorectal cancer remained unknown, and has not been reported at home and abroad.Integrin belongs to the family of cell surface adhesion molecules, and is a kind of transmembrane glycoprotein receptors combined with alpha and beta subunits in a covalent bond, mediating the adhesion between cells and cells or cells and extracellular matrix, promoting cell proliferation, differentiation, invasion and metastasis via affecting the cell gene expression and cell signal transduction inside or outside.Integrinαvβ6 is the only special integrin subtypes induced expressing in malignant epithelial tumors but not in normal and benign tumor tissue. Integrinαvβ6 is highly expressed in the process of embryonic development and in the glandular epithelium of digestive tract tumor such as colorectal cancer, gastric cancer, pancreatic cancer and bile duct carcinoma; Integrin αvβ6 is also found expressing in squamous epithelial tumor such as lung cancer, squamous cell carcinoma of the oral; Integrin αvβ6 is mainly expressed in the edge of invasive tumor involved in a variety of tumor malignant behavior such as proliferation, differentiation, invasion, migration and resistance to apoptosis. Our previous studies have shown that:The expression of αvβ6 is different in different types of colonrectal cancer cells; Integrin αvβ6 is highly expressed in the invasive edge of colon cancer specimens, and lower expressed within the tumor, which is closely associated with its malignant progression.This topic intends to observing the expression changes of integrin αvβ6 in these colorectal cancer cells after radiotherapy, and to explore the specific molecular mechanism of integrin αvβ6 in mediating malignant behavior such as invasion and apoptosis resistance in these colorectal cancer cells on the basis of our previous studies. further understanding the serious perniciousness of radiotherapy resistance and the role of integrin αvβ6 in regulating the malignant progress of colorectal cancer. All these help to make integrin αvβ6 as a target for efficient non-toxic therapy assisting the radiotherapy of colorectal cancer, which playing an important role in providing experimental evidence and theoretical basis for improving the radiotherapy curative effect of the colorectal cancer and promoting the prognosis.PartⅠEpieriocalyxin A induces cell apoptosis through JNK and ERK1/2 signalling pathways in colon cancer cell lineObjectiveInvestigate the pharmaco-mechanism of Epieriocalyxin A induce colonrectal cancer cell apoptosis.Methods1. Cell proliferation assay was performed using Cell counting kit-8 (CCK8) assay after Caco-2 cells were treated with 0,0.2,0.5 and 1μg/ml of EpiA (Epieriocalyxin A) for 0,12,24,48, and 72 hr.2. Annexin V assay was performed to examined the flopped annexin-V level in the surface of cells and determine cell apoptosis in Caco-2 cells treated with 0, 0.2,0.5 and 1 μg/ml of EpiA for 24h.3. Hoeehst 33342 staining assay was performed to observe the Cell apoptosis and DNA fragment in Caco-2 cells treated with 0,0.2,0.5 and 1 μg/ml of EpiA.4. ROS assessment assay was performed to examine the ROS in Caco-2 cells treated with EpiA after 24h.5. Mitochondrial membrane potential assay kit with TMRM was performed to examine the mitochondria membrane potential and mitochondria damage in Caco-2 cells treated with EpiA.6. SDS-PAGE and Western blot assay was performed to analyze the expression and phosphorylation degree of phosphatidylinositol 3-kinase (PI3K) and Mitogen Activated Protein Kinases (MAPKs) in the Caco-2 treated by EpiA.7. Western blot assay was further performed to analyze some apoptosis related proteins expression such as capsase 3, Bcl-2 and Bax.8. Hoechst 33342 staining was used to detect apoptosis when ROS induced by 1 μg/ml EpiA was inhibited by antioxidants NAC. Apoptosis was detected when JNK and ERK1/2 pathway was blocked by SP600125 and PD98059 separately.9. Western blot was used to detect the expression of p-JNK and p-ERK1/2 when ROS was inhibited by antioxidants NAC in Caco-2 cells treated with 1 μg/ml EpiA.10. Caspase-3, Bcl-2 and Bax when ROS was inhibited by antioxidants NAC which induced.by 1μg/ml EpiA. Protein expression was detected when JNK and ERK1/2 pathway was blocked by SP600125 and PD98059 in Caco-2 cells.11. Western blot was used to detect protein expression of pIκcBα and cytochrome C in Caco-2 cells treated by EpiA, or EpiA combined with NAC,or SP600125 and PD98059.Results1. EpiA cell significantly restrain the Caco-2 colon Cancer cells growing, and this kind of inhibition effect is positive correlation with EpiA dose and treatment time.2. EpiA can induce Caco-2 colonrectal cancer cells apoptosis, using 1μg/ml EpiA treat cell for 24 hours, the apoptosis rate is 41.6%. Annexin V assay show that Caco-2 cells are in the early stage of apoptosis in the presence of EpiA.3. Hoeehst 33342 staining assay showed that when the EpiA concentration increased to 0.5 μg/ml, nucleus DNA starts to be condensed, and after treated with 1 pg/mlof EpiA, nucleus DNA is fragmented.4. ROS assessment assay confimed that EpiA treatment could considerably induce the generation of ROS in Caco-2 cells.5. Mitochondrial membrane potential assay kit with TMRM showed that EpiA could induce the falling of Mitochondrial membrane potential and a mitochondria-mediated cell apoptosis.6. SDS-PAGE and Western blot assay showed that JNK phosphorylation was gradually decreased in a dose response with EpiA. The Western blot results showed that ERK1/2 phosphorylation is also reduced in the presence of EpiA. However, we did not observe any change of P38 phosphorylation level.7. SDS-PAGE and Western blot assay further confirmed that EpiA treatment upregulated the expression of capsase 3 and reduced expression of Bcl-2 in a dose response manner. In contrast, the Bcl-2 negative regulator, Bax, showed an increased expression.8. Hoeehst33342 staining showed that there is no significant change of cell apoptosis rate in NAC group or when compared with Oμg/ml EpiA treated group, however, the apoptosis rate was significantly decreased when compared with 1μg/ml EpiA treated group. SP600125 (JNK inhibitor) could induce apoptosis in Caco-2 cells, and the apoptosis rate was higher than Oμg/ml EpiA treated group and significantly lower than 1μg/ml EpiA treated group; PD98059 (ERK1/2 inhibitor) could induce apoptosis in Caco-2 cells, and the apoptosis rate was higher than Oμg/ml EpiA treated group and significantly lower than 1 μg/ml EpiA treated group.9. Western blot results showed that there is no significant change of phospho-JNK (p-JNK) and phosphorylation-ERK1/2 (p-ERK1/2) in Caco-2 cells treated with 1μg/mlEpiA and NAC, that is the (1+NAC) group, when compared with Oμg/ml EpiA treated group. However, the expression of p-JNK. and p-ERK1/2 were significant higher than that of 1 νg/ml EpiA treated group.10. Western blot assay further confirmed that upregulation expression of caspase-3, Bax and downregulation of Bcl-2 induced by 1 μg/ml EpiA were significantly inhibited when combined with antioxidants NAC. Furthermore, there is no significant change in the protein expression of caspase-3, Bax and Bcl-2 between (1+NAC) group and 1μg/ml EpiA treated group.The induction of Caco-2 cells to EpiA were completely reversed. SP600125 (JNK inhibitor) and PD98059 (ERK1/2 inhibitor) both could induce upregulation expression of caspase-3, Bax and downregulation of Bcl-2 in Caco-2 cells, and the increase of caspase-3, Bax was higher than that of Oμg/ml EpiA treated group and significantly lower than that of 1μg/ml EpiA treated group, and the decrease of Bax was also higher than that of Oμg/ml EpiA treated group and significantly lower than that of 1 μg/ml EpiA treated group.11. Western blot assay confirmed that EpiA treatment downregulated the expression of pIκBα and upregulated the expression of cytochrome C in a dose response manner. The downregulation expression of pIκBα induced by were counter-balanced when combined with antioxidants NAC.The combination of SP600125 and PD98059 could significantly inhibit the expression of pIκBα.There was no significant difference between 1μg/ml EpiA-treated group and SP600125 and PD98059 treated group.Conclusion and SignificanceIn this study, we showed that Epieriocalyxin A (EpiA), a diterpenoid isolated from Isodon erioclyx, could suppress the growth of Caco-2 colon cancer cells. EpiA could induce annexin-V flipping in cell membrane and DNA fragment. We also showed that EpiA could induce the generation of ROS in Caco-2 cells, as well as the damage of mitochondrial membrane. We further detected cell signaling pathways involved in these processes induced by EpiA using Western blot. Both JNK and ERK1/2 activation were decreased after treated with EpiA in a dose response manner. EpiA could increase the expression of caspase 3 and Bax, and decrease Bcl-2 expression. We further confirmed that cell apoptosis induced by EpiA could be completely reversed by antioxidants NAC upon ROS via the inhibition of upregulation of caspase-3, Bax and downregulation of Bcl-2 partly mediated through JNK and ERK1/2 signaling transduction pathway. The blockade of JNK and ERK1/2 signaling could account for the inhibition of pIκcBα which affact NF-kB and its downstream transcription of Bcl-2. Thus, PTP was open and cytochrome c was released and thereby mitochondrial apoptosis pathway was promoted. Our results suggest that EpiA is a novel compound that induces colonrectal cancer apoptosis. EpiA could be a potential drug agent for colonrectal cancer therapy in the future.PartⅡUpregulation of β6 integrin and its role in ionizing radiation enhanced invasiveness and apoptosis resistance of colorectal cancer cellsObjectiveInvestigate the specific molecular mechanism of (β6 integrin in regulating malignant progression and radiotherapy resistance in colorectal cancer.Methods1. Transwell invasion assay was used to detect the invasion ability of colonrectal cancer cells HT-29, WiDr and Caco-2 subjected to radiotherapy treatment with irradiation treatment of OGy,2Gy,4Gy,6Gy,8Gy(IR group) and without irradiation treatment in vitro (Non IR group); Invasion ability of post-IR HT-29, WiDr and Caco-2 were observed at 6h、8h、16h、24h.2. RT-PCR and Western blot assay was used to detect mRNA and protein expression of integrin αv and β6 subunit in HT-29, WiDr and Caco-2; RT-PCR and Western blot assay was used to detect mRNA and protein expression in HT-29 subjected to gradient irradiatin of OGy,2Gy,4Gy,6Gy,8Gy.3. Three synthetic double chain with different sequences of small interfering RNA (small interfering RNA, siRNA)were transfected into colonrectal cancer cells HT-29 by liposome 2000 with importing deleted. Then after 24h RT-PCR assay was used to detecting the level of β6 mRNA expression, the effective siRNA sequences which could restrain integrin β6 expression were screened out. Transwell invasion assay was used to detect invasion ability of HT-29 before and after 6Gy IR in groups (1) Control (2) AIIB2 (3) LM609 (4) F1P6 (5) IgGl (6) 10D5 (7) IgG2a. Transwell invasion assay was used to detect invasion ability of HT-29 before and after 6GyIR in groups (1) Control组 (2) siβ6 (3) siNC (4) 10D5 (5) IgG2a (6) PD98059.4. Western blot assay was used to detect protein expression of MMP-2 and MMP-9 in HT-29 subjected to gradient irradiatin of OGy,2Gy,4Gy,6Gy,8Gy; Western blot assay was used to detect protein expression of MMP-2 and MMP-9 in HT-29 (1) Non IR (2) IR (3) IR+siβ6 (4) IR+siNC (5) IR+IgG2a (6) IR+10D5, and the dose of IR was 6Gy.5. Western blot assay was used to detect protein expression of key factors such as ERK, P-ERK, JNK, P-JNK, P38, P-38 in MAPK signaling pathways in HT-29 subjected to 0Gy,2Gy,6Gy; Western blot assay was used to detect protein expression of ERK, P-ERK, JNK, P-JNK, P38, P-38 in (1) Non IR (2) IR (3) IR+sip6 (4) IR+10D5 (5) IR+siNC (6) IR+IgG2a, and the dose of IR was 6Gy.6. Nude were divided into four groups(1) Non IR (2) IR (3) IR+10D5(a functional monoclonal blocking antibody against αvβ6) (4) IR+IgG isotype.The same amount of colonrectal cancer cells HT-29 were injected subcutaneously into nudes, and When the xenografts reached a mean diameter 0.8-1 cm, a peritumoral injection of 10D5 or IgG isotype were also administrated. All radiationtherapy groups were exposed to 20Gy fractionated irradiation. The tumorigenic capacity of the xenografts was excised and the volume was measured and calculated three weeks after treatment, and TUNEL immunohistochemical staining was used to detect apoptosis.7. Caco-2 colonrectal cancer cells were divided into three groups:Mock-transfectant, β6-transfectant and P6 Mutant-transfectant(in which β6-integrin lost the ERK2 binding site). Western blot assay was used to detect protein expression of MMP-9, Bcl-2 and Bax in post-6GyIR HT-29 cells.Results1. Transwell invasion assay results confirmed that IR significantly promote the invasion ability of HT-29 and WiDr cells, and IR has no significant effect on invasion ability of Caco-2 cells; Invasion ability of post-6GyIR HT-29 and WiDr cells were increased at 6h、8h、16h、24h.2. RT-PCR and Western blot results showed that HT-29, WiDr have positive expression of integrin 06, and Caco-2 cells do not express integrin 06, and αv integrin expression have no significant difference in three groups of cells. IR could significantly increase mRNA and protein expression of integrin β6 in colonrectal cancer cells HT-29 and WiDr, and the peak expression was observed when subjected to 6GyIR. IR has no effect on the expression of integrin av in cancer cells HT-29.3. Transwell invasion assay showed that 10D5, a functional monoclonal blocking antibody against αvβ6 inhibit invasion ability of HT-29 (P<0.05),and when it combined with IR this inhibition was significantly amplified(P<0.01). Invasion ability has no significant change in HT-29 cells when treated with monoclonal blocking antibody AIIB2(against αvβ1), LM609(against αvβ3) and F1P6(against αvβ5)(P>0.05); Inhibition of integrin β6 mediated by siRNA significantly inhibit invasion ability of HT-29 when subjected to 6GyIR. Specific ERK signaling pathway inhibitor PD98059 can obviously inhibit invasion ability in post-6Gy IR HT-29 cells(P<0.05).4. Western blot assay confirmed that gradient IR of OGy,2Gy,4Gy,6Gy,8Gy could upregulate protein expression of MMP-9 in HT-29(P<0.05 vs Non IR), and has no significant effect on MMP-2(P>0.05 vs Non IR); β6 gene silence mediated by siRNA and 10D5 significantly inhibit the expression of MMP-9 (P<0.05 vs Non IR). PD98059 can also inhibit the expression of MMP-9 in post-6Gy IR HT-29 cells (P<0.05 vs Non IR)and has no effect on MMP-2 (P>0.05)5. Western blot results showed that irradiation could enhance the p-ERK1/2 expression in HT-29 cells(P<0.05 vs Non IR), but had no effect on the the expression of t-ERK1/2, p-JNK, p-p38, t-JNK and t-p38 (P>0.05).β6 gene silenced by specific siRNA or blocked by 10D5, could inhibit the upregulation expression of p-ERK1/2 (P<0.05 vs Non IR)and had no effect on the expression of P-JNK and P-38 (P>0.05) in 6Gy pretreated HT-29 cells.6. αvβ6 integrin blockade by 10D5 synergistically inhibitted the growth of tumor and increased the effect of irradiation on xenografts. The average volume of Non IR tumors was about (361±17.23) mm3, IR group (284±14.97) mm3, (IR+10D5) group (61±7.79)mm3 and (IR+IgG isotype) group (265±13.74) mm3. The average volume of (IR+10D5) tumor was significantly smaller than Non IR group and IR group(P<0.05). TUNEL results showed that the apoptotic rate of cells in IR+10D5 group was significantly higher than that of IR or Non IR group (P<0.05).7. Western blot results showed that β6-integrin-mediated Bcl-2 up-regulation, MMP-9 up-regulation and Bax down-regulation in post-IR HT-29 cells, were also inhibited when β6-integrin lost the ERK2 binding site(P<0.05).Conclusion and SignificanceThis research revealed that IR could upregulate integrin β6 expression in colorectal cancer cells, and integrin β6 mediate extracellular matrix degradation and invasion induced by IR through ERK/MAPK signaling pathways. Deleting the ERK2 binding site could downregulate Bcl-2, MMP-9 expression and upregulate Bax expression, restraining invasive growth and radioresistance which induced by IR and mediated by β6-integrin. Thus,β6 blockade synergistically induced colonrectal cancer cells HT-29 apoptosis, and inhibited the growth of its xenografts. This research unfolded the expression changes of integrin β6 in CRC cells insulted by irradiation; further disclosed the important role of integrin β6 and β6-ERK2 direct linkage in regulating the malignant biological behaviour of CRC cells exposed to irradiation; enriched basic knowledge of the integrin β6 and provided the precise theory for future treatment targeting β6 in clinical study field of sensitizing radiotherapy. |
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