Leucovorin Enhances The Anti-cancer Effect Of Bortezomib In Colorectal Cancer Cells

Backgroud:Colorectal cancer(CRC),is the development of cancer from the colon or rectum.Globally,colorectal cancer is the third most common type of cancer making up about 10% of all cases.It is more common in developed countries,where more than 65% of cases are found.It is less common in women than men.CRC is the abnormal growth of cells that have the ability to invade or spread to other parts of the body.Signs and symptoms may include blood in the stool,a change in bowel movements,weight loss,and feeling tired all the time.Most colorectal cancers are due to old age and lifestyle factors with only a small number of cases due to underlying genetic disorders.Dietary factors that increase the risk include red and processed meat as well as alcohol.Another risk factor is inflammatory bowel disease,which includes Crohn's disease and ulcerative colitis.It typically starts as a benign tumor,often in the form of a polyp,which over time becomes cancerous.Treatments used for colorectal cancer may include some combination of surgery,radiation therapy,chemotherapy and targeted therapy.Five year survival rates in the United States are around 65%.This,however,depends on how advanced the cancer is,whether or not all the cancer can be removed with surgery,and the person's overall health.The standard cytotoxic chemotherapy regimen for CRC patients is 5-fluorouracil(5-FU)with leucovorin plus either oxaliplatin(FOLFOX)or irinotecan(FOLFIRI).Despite initial clinical response,a large portion of CRCs develops resistance to 5-FU.Therefore,there is an unmet clinical need to explore novel therapeutic agents or new combination treatments to achieve CRC remission.Objective:In this study,we performed a novel drug-repurposing screening to identify Food and Drug Administration-approved chemotherapeutic compounds possessing synergistic activity with leucovorin against CRC cells.Methods:1.Screening of the Approved Oncology Drugs Set VII from NCI.cell viability as determined by MTT assays.2.Cell Titer-Glo assays: percentages of cell viability of HCT116 and HT29 cells upon treatment with combination of bortezomib(BTZ,3 n M~300 n M)and leucovorin(LV,10 ?M)were compared to those treated with bortezomib alone for 24 h and 48 h.3.Flow cytometry detect apoptosis: HCT116 and HT29 cells were treated with combination of bortezomib(3 or 10 n M)and leucovorin(10 ?M)or either drug alone for 12 h and 24 h,respectively.The treated cells were stained with FITC-Annexin V/propidium iodide.4.Western blot: detect the protein expression of caspase family,PARP,Bcl-2,p53,p-p53,H2 AX,PUMA,Bax,p21,pJNK,p-ERK1 / 2(Thr202 / Tyr204),p-Akt and p-Bcl-x L.5.CRC xenografts in mice: HCT116 cells(5x105 per mouse)were subcutaneously injected into the right flanks of NSG mice.When the tumors reach 100 mm3,mice(n=5 per group)were randomized into four groups for treatment with vehicle,0.5mg/kg bortezomib,80mg/kg leucovorin,or the combination twice a week for 4 weeks by intraperitoneal injection.6.TUNEL assay: sections of paraffin-embedded tissues were deparaffinized then washed with TBS buffer and permeabilized with proteinase K.DNA strand breaks were then end-labeled with terminal transferase,and the labeled DNA was visualized by fluorescence microscopy.Results:We found that the combination of bortezomib and leucovorin enhanced caspase activation and increased apoptosis in CRC cells better than either agent alone.Further,the synergistic induction of apoptosis and inhibition of tumor growth were also observed in mouse CRC xenografts.Conclusion:Our data call a new clinical trial to evaluate the combinatorial treatment with bortezomib and leucovorin against metastatic CRC.