The Studies Of Intgrin αvβ6 Regulate Colon Cancer Cell Malignant Biological Behavior

Background:Colon cancer is one of the most frequent digestive tract cancers. It's incidence rate is elevating year by year with the economy raising and the changes of people's food. The traditional surgical operation is the best method to treat the colon cancer, but most patients are on the middle-advanced stage and have missed the chance of accepting the operation. Hence, chemotherapy has already been the first adjuvant therapy to the advanced stage colon cancer. Unfortunately, chemotherapy will produce severe side reaction, patients cannot be tolerant. It is a research heat-point in colon cancer treatment in current and future that how to decrease the adverse reaction with the premise of good treatment effectiveness. On this background, the traditional Chinese medicine has been through highly because they have many good characteristics, such as stronger anti-tumor effects, lighter adverse reaction and easy to be tolerant.Norcantharidin (NCTD) is the extract of cantharis. Many domestic hospitals have proven that taking the NCTD with the classical chemotherapy in colon cancer treatment is better than only thermotherapy. Comprehensive treatment has many good qualities, for example, better short-term curative effect, lighter adverse reaction, longer meta-live time, better life quality. NCTD has been the best adjuvant drug in first line thermotherapy of colon cancer treatment. However, the mechanism of NCTD anti-tumor is unclear. There are many explanations to its mechanism, but no decisive conclusion.Integrin is one kind of cell adhesion factors. They are composed byα&βsubunits which are linked through non-covalent bond. Integrin is the bridge between extra-cell matrix and intra-cellular skeleton protein.αvβ6 is a special isoform of integrin. They do not or almost not express in epithelial tissue of health people. But they highly express in embryogenesis, trauma repairing and a series of malignant epithelial tumors. Now, the researches about integrinαvβ6 how regulate the malignant tumor biological behavior have gotten plentiful results, especially in the researches on colon cancer. Some paper has been reported that integrinαvβ6 could promote colon cancer cell proliferate, invasive, resisting apoptosis and accelerate tumor cell form cancer. Our previous research has already proven that NCTD could restrain colon cancer express the integrin avP6. This research (part 1) is focuing on investigating the specific mechanism of NCTD inhibits colon cancer growth, and developing new target drugs according to the pharmaco-mechanism.For many years, about the original question of tumor, there are two theories in medical field. Stochastical theory presumes that all of tumor cells are like each other in shape, size and volume. Any one of tumor cells can be differentiated and form a new tumor, but it is some low probability random event. Hierarchy theory deems that only an exceeding small amount cell can be differentiated and form a new tumor. Those cells which can be differentiated and form a new tumor are called cancer stem cell. They have the ability self-renewal ability and differentiation, and are the source of tumor cell keep growing and migrating. Recently, the cancer stem cell theory acquires more and more study results to support. Colon cancer stem cell is the first discovered digestive tract malignant tumor stem cell.Based on the cancer stem cell theory, stem cell continuing existing is tightly related to tumor malignant extent level and prognosis. Most cancer stem cell is on the G0/G1 resting phase, chemotherapy survivability of those stem cells is obviously stronger than quick proliferative phase tumor cells. Special form of new tumor capability is the main reason of malignant tumor recurrence and metastasis. Combining our previous research results in histology, cytology and animal experiments, we believe that the characteristic ofαvβ6 expressing colon cancer cell including resisting apoptosis, proliferation, invasion, and tumor forming, are coincide with those characteristic of cancer stem cell including strong drug survivability, infinity proliferation, and easily tumor forming. This research (part 2) focus on investigating the expression of integrin avP6 on colon cancer stem cells, and identifying the possibility of integrinαvβ6 to become the colon cancer stem cell molecular marker and establish a substantial base for future research, including sorting, identifying, and lucubrating those cancer stem cell.PartⅠNorcantharidin induces HT-29 colon cancer cell apoptosis through theαvβ6-ERK signaling pathwayObjectiveInvestigate the pharmaco-mechanism of Norcantharidin (NCTD) restrain colon cancer cell growing.MethodsMTT assay was used to detect the inhibition effect of NCTD on HT-29 colon cancer cell growth. The matrix metalloproteinase (MMPs) content secreted by HT-29 cell treated by NCTD was detected by the gelatin zymography assay. Biotrak MMPs activity assay system was used to inspect the MMPs activity changing when HT-29 cell treated by NCTD. Hoechst 33258 fluorescent staining was used to sense the degree of HT-29 colon cancer cells apoptosis. Flow cytometry was used to detect some kinds of integrin expression variance produced by NCTD. MTT assay was used again for detecting the growth inhibition effects of several functional blocking antibodies on NCTD treated HT-29 colon cancer cells. Western Blotting assay analyze the expression and phosphorylation degree of Mitogen Activated Protein Kinases (MAPKs) in the HT-29 colon cancer cell treated by NCTD. MTT assay was used to detect the growth inhibition effects of several MAPKs inhibitors on NCTD treated HT-29 colon cancer cells. Co-immunoprecipitation assay was used for detecting the effect onαvβ6-ERK direct linkage produced by NCTD.ResultsNCTD can obviously restrain the HT-29 colon cancer cells growing, and this kind of inhibition effect is positive correlation with NCTD dose and treatment time. NCTD can also restrain the HT-29 colon cancer cells secrete the MMP-9 and its activity, but no inhibition effect on MMP-3. NCTD can induce HT-29 colon cancer cells apoptosis, using 60μmol/L NCTD treat cell for 12 hours, the apoptosis rate is 35.6%. Flow cytometry assay show that NCTD can restrain the HT-29 colon cancer cells expresses the integrinαvβ6, but the expression ofαvβ3 andαvβ5 were not affected. The Western Blotting assay further confirmed that only the content of (36 subunit decreased obviously, the levels ofαv,β3 andβ5 subunits did not change. MTT assay show only 10D5 (a functional blocking antibody toαvβ6) can strengthen growth inhibition effect on HT-29 colon cancer cells produced by NCTD, LM609 (functional blocking antibody toαvβ3) and P1F6 (functional blocking antibody toαvβ5) cannot. Western Blotting analysis showed, only the content of phosphorylate-ERK (p-ERK) decreased accompanying with the NCTD dose richer or treatment time longer. But the levels of ERK, JNK, p-JNK, P-38 and p-P38 did not change. The inhibitor of ERK PD98059 can inhibit HT-29 colon cancer cells grow, but both the SP600125 (JNK inhibitor) and SB 203580 (P38 inhibitor) cannot. Co-immunoprecipitation assay certified that the formation of avP6-ERK direct linkage was disturbed by NCTD.ConclusionNCTD interfere with the phosphorylation of ERK because it decreases the expression of integrinαvβ6. Hence, the direct linkage betweenαvβ6 and ERK was broken, and the signal mediated byαvβ6 was blocked. Thus, NCTD induce the HT-29 colon cancer cells apoptosis, and inhibit the cancer cells growing.Significance This research disclosed the mechanism of NCTD induce colon cancer cell apoptosis and inhibit cancer cells growth; further confirmed the important role of integrin av(36 andαvβ6-ERK direct linkage in regulating the malignant biological behavior of cancer cells; provided the precise theory for future target treatment toαvβ6 andαvβ6-ERK direct linkage in colon cancer treatment.PartⅡThe Role of Integrinαvβ6 in Regulating Colon Cancer Stem Cell differentiates to powerful invasive colon cancer cellObjectiveIdentify the relationship between the integrinαvβ6 expression and colon cancer stem cell, and investigate the role of integrinαvβ6 in regulating the colon cancer stem cell malignant biological behavior.MethodsImmunohistochemisty staining analyzes the expression correlation between the integrinαvβ6 and CD133 in colon cancer tumor tissue. FACScan were carried out to detect the percentage of CD133+ cell in HT-29 colon cancer cell, and sorted those CD133+ cell with its mAb ANC9C5. Cultured those CD133+ cell in serum-free medium, and observed cancer stem cell morphocytology characteristic. Using siRNA technology to interfere with the expression of avP6 followed MTT assay was carried out to inspect if the avP6 expression can affect the colon cancer stem cell growth. Zymogram analysis was used to detect the effect ofαvβ6 expression on colon cancer cell MMP-9 secretion.ResultsImmunohistochemisty staining confirmed that the expression ofαvβ6 and CD 133 are consistency, and both of them are prone to highly express in the leading edge of tumor tissue. The FACScan analysis showed that the percentage of CD133+ cell in HT-29 colon cancer stem cell was 1.6%. After Sorting CD133+ cell by FACScan with mAb ANC9C5, the percentage increased to 92.6%. Cultured those CD133+ cell in serum-free medium, those cell can form several small tumor cell globes in 72 hours, present classical stem cell characteristic. Added fetal calf serum into serum-free medium, tumor cell globes disappear gradually, cancer cell grow adherence again, showed out the differentiated characteristic. Constructed anti-β6shRNA plasmid, and transduced the plasmid into CD133+ cell, followed by RT-PCR assay and Western Blotting analysis. Both of the two experiments conformed that the mRNA and protein levels of av(36 decreased obviously. MTT assay showed that only theαvβ6+ CD133+ HT-29 colon cancer cells can normally divide and proliferate in serum-free medium, but the avP6 CD133+ HT-29 cannot. Gelatinase zymogram analysis also confirmed that MMP-9 secreted amount ofαvβ6+ CD133+ HT-29 cell were obviously more thanαvβ6- CD133+ HT-29 colon cancer cell.ConclusionIntegrin avP6 and CD133 express in same period and same area in colon cancer tumor tissue. Integrinαvβ6 can promote CD133+ HT-29 colon cancer cell proliferation, and induce them secrete MMP-9. Integrinαvβ6 play an important role in regulating colon cancer stem cell differentiates to powerful invasive colon cancer cell.SignificanceDisclosed the important role of integrinαvβ6 in regulating colon cancer stem cell differentiates to powerful invasive colon cancer cell. Because tumor surrounding tissues enrich powerful invasive tumor cells, integrin avP6 also should be deemed as a molecular marker of invasive colon cancer stem cell.