The Inhibitory Effects Of Bortezomib On Colon Cancer Cells And The Underlying Mechanisms

Background: Colon Cancer is the third most common cancer in the world and is one ofthe nine most common cancers in China. Social environment, lifestyle and genetic factorsare associated with colon cancer risk.The morbidity of colon cancer had been increased inthe past20years and will be continuing.Colon cancer can also cause many seriouscomplications, such as intestinal obstruction, intestinal perforation and others. Althoughmany advances have been achieved in chemotherapy and surgery treatment, the5-yearsurvival rate and life quality in late stage of patients are still not satisfactory. Therefore,new types of drug therapy or combination of current therapies are needed in the treatmentof colon cancer to reduce recurrence and metastasis after late surgery and to improve thesurvival rate and life quality of patients with colon cancer. Bortezomib is the firstproteasome inhibitor used in clinic. In2003, it was approved by FDA for the treatment ofmultiple myeloma. Bortezomib was found to potently inhibit cell proliferation in astandard National Cancer Institute screen of60cell lines derived from human tumors.Some studies suggested that bortezomib took its effect through the nuclear factor kappa B(NF-kappa B) pathway while other found PI3K/Akt pathway was crucial. Nowadays, bortezomib-induced cell growth inhibition and apoptosis induction were observed in vitroin many different kinds of malignant cells including multiple myeloma, prostate cancer,pancreatic cancer, renal cell carcinoma, and squamous cell carcinoma, whether it hastherapeutic potential in colon cancer is still unknown.Objective:1. To observe the effect of bortezomib on the malignant behaviors of coloncancer cell line HT-29；2. Explore the mechanism of bortezomib that influence biological behaviorsof the colon cancer by PTEN-PI3K/Akt pathway.Method:1. Human colon cancer cell HT-29was routinely cultured in vitro and treatedwith different concentrations of bortezomib (1-500nmol/L) for24-48h.2.The changes ofcell growth and apoptosis were investigated by MTT assay and Annexin-V/PI doubledyeing assay.3. The effects of bortezomib (80nmol/L) on migration and invasion abilitiesof HT-29cells were investigated by wound healing assay and invasion assay respectively.4. The subcellular location of E-cadherin was detected using a laser scanning confocalmicroscope.5. The protein level of cleavage of caspase-3, E-cadherin, phosphor-Akt andPTEN were determined by Western bolt.6.Recombinant lentiviral vectors encodingshRNAs were designed to down-regulate PTEN expression in HT-29cells. Then harvestedstable PTEN knockdown HT-29cells and observed the change of invasion and apoptosisin cell after treatment with bortezomib.Results:1.Bortezomib significantly inhibited the proliferation of HT-29cells in a dose-and time-dependent manner. The IC50value at48h was82.43nmol/L.2.Treatment with80nmol/L of bortezomib significantly induced apoptosis at24h, and reached a peak at48h. HT-29cell cycle arrested at G0/G1phase after treatment with bortezomib for24h.3.Bortezomib could effectively reduce the migration and invasion of HT-29cells bywound healing assay and invasion assay.4. It was found that cell membrane andcytoplasm transporter appeared in cells treated with80nmol/L of bortezomib by a laserscanning confocal microscope.5. The protein level of PTEN,E-cadherin was obviouslyup-regulated while phosphor-Akt level was down-regulated in HT-29cells treated with80nmol/L of bortezomib by Western Blot.6.Recombinant lentiviral vectors encoding shRNAs were designed to down-regulate PTEN expression in HT-29cells. We harvestedstable PTEN knockdown HT-29cells, and observed the change of invasion and apoptosisin cells after treatment with bortezomib. There was no significant difference. The proteinlevel of PTEN,phosphor-Akt was obviously by Western Blot.Conclusion: Bortezomib could inhibit the proliferation of colon cancer HT-29cells,induced cell cycle arrest and apoptosis, and decreased cell migration and invasion ofability. We propose that bortezomib could effectively affect the PI3K/Akt signalingpathway in human colon cancer HT-29cells by suppressing the degradation of PTEN.These findings suggest that bortezomib may hold a therapeutic promise in thechemotherapy of colon cancer.