Combination Of Salinomycin And Gemcitabine Eliminates Pancreatic Cancer Cell

Background and ObjectivePancreatic cancer is the fourth most common cause of cancer death. Despite significant improvements in diagnostic imaging and operative mortality rates during the past two decades, the5-year survival rate remains lower than5%. Gemcitabine (GEM) has been the most commonly used chemotherapeutic agent over the past decade. Unfortunately, numerous phase â…¢ trials testing GEM combined with other cytotoxic drugs have failed to reveal any additional benefit compared with GEM alone. Emerging data suggest that malignant tumors are composed of a small subset of distinct cells termed cancer stem cells (CSCs), which are responsible for tumor initiation and propagation. Over the past few years CSCs have been discovered in several types of solid tumors, including pancreatic cancer. Most current systemic therapies have been found ineffective in the treatment of solid tumors, and this may be due, at least in part, to the increased resistance of CSCs. Therefore, it is importance to discover new therapeutic modalities to eliminate CSCs in order to eventually develop protocols for more successful treatment.Salinomycin (SAL) was originally used to kill bacteria, fungi, and parasites and fed to ruminants to improve nutrient absorption and feeding efficiency. However, SAL has recently been shown to selectively deplete human breast cancer stem cells. Thus, SAL may be a promising chemical for eradication of CSCs. Here we investigated whether SAL alone or combined with GEM was capable of eliminating pancreatic cancer cells both in vitro and in vivo.Methods(1) Cytotoxic effects of SAL and GEM on pancreatic cancer cells separated by FACS in vitro1. Separation of CD133+/CD133-and SP/NSP cells by FACS from human pancreatic cell lines SW1990and AsPC-1.2. We evaluated the antiproliferative effects of SAL, GEM and combination using MTT assays on both CD133+/CD133-and SP/NSP cells isolated from the SW1990and AsPC-1cell lines.3. We used an Annexin V-FITC assay to test the extent of apoptosis in both CD133+and CD133-cells isolated from the SW1990and AsPC-1cell lines following72hours of treatment with SAL, GEM, or their combination.;4. We exposed SW1990cells isolated by FACS to GEM, SAL, or combined treatment for72hours. And evaluate whether single or combined treatment could suppress the formation of colonies in vitro. To test the self-renewal feature further, we performed the sphere-forming assay after drug treatment.(2) Regression of human pancreatic cancer xenografts following combined treatmentWe subcutaneously inoculated CD133+or CD133-cells isolated from the SW1990cell line into the flank of nude mice. Once the tumors reached a mean diameter of2-4mm, both groups of mice (CD133+and CD133-) were randomized to four subgroups with six animals each. The mice were then treated with PBS, GEM, SAL, or their combination. GEM was administered twice a week (100mg/kg), while SAL was administered every other day (4mg/kg), both were injected intraperitoneally over25days.Results1. SW1990and AsPC-1cell lines could separate CSCs by FACS, in which the proportion of either CD133+or SP cells were relatively high.2. With increasing concentrations, the survival rate of CD133+cells treated with SAL decreased more sharply than that of CD133+cells treated with GEM or CD133-cells treated with SAL, whereas the cytotoxic effects of GEM on CD133-cells was significantly better. Similarly, the viability of SP cells treated with SAL was inhibited more strongly compared with that of SP cells treated with GEM or NSP cells treated with SAL, including cells from both the SW1990and AsPC-1cell lines3. Cell growth inhibition was determined by MTT assay in the SW1990and AsPC-1cell lines. Combined treatment of SAL and GEM could eradicate both CD133+and CD133-cells more efficiently than either SAL or GEM alone (P<0.01). Likewise, we performed the same assay on SP and NSP cells and observed similar findings.4. SAL alone could inhibit the formation of CD133+cell colonies and sphere, but showed a lower efficiency on CD133-cells. For combined treatment, either CD133+or CD133-cells could effectively form colonies (both P<0.01).5. In the CD133+group, administration of GEM or SAL alone retarded tumor growth, but the combined treatment of GEM and SAL almost completely inhibit tumor growth (P<0.01vs. GEM or SAL group). Either GEM alone or the combined treatment inhibited the tumor growth of CD133-cells significantly (P<0.01vs. SAL treatment). Importantly, there was no significant change in the body weight of the mice.ConclusionsOur pervious data indicate that SAL can inhibit pancreatic CSCs. More importantly, our current results indicated combined treatment of SAL and GEM eliminated both CSCs and differentiated cells. As such, SAL could be a promising agent for novel combination therapy for the treatment of human pancreatic cancers.