e14131 Background: A small subpopulation of chemotherapy resistant cancer stem cells (CSCs) drive majority of epithelial cancers including colon cancer. We have observed that chemosurviving (cells that survive acute chemotherapy insult) colon cancer cells are enriched in CSCs and express increased levels of IGF-1R (4.5-fold) (Transl Oncol. 2009;2:321). We hypothesized that depletion of IGF-1R levels by si-RNA would result in growth inhibition of cancer stem-like cells and restore growth inhibition mediated by chemotherapy. Methods: Chemosurviving HCT-116 (p53 wild type), HCT-116 p53-/- and HT-29 (p53 mutant) colon cancer cells were grown in DMEM media in the presence of oxaliplatin and 5-fluorouracil (5-FU). Western blot and quantitative RT-PCR were used for CSC marker expression. Cancer specific microRNA array (Invitrogen) and TaqMan Q-RT-PCR for miR levels were performed according to manufacturer's instructions. Results: Depletion of IGF-1R by si-RNA resulted in 35% inhibition of colonosphere formation, a CSC property, accompanied by a reduction in CD44 and CD166 levels particularly in p53 wild type (HCT-116) colon cancer cells. On miR array, we identified miR-215, a p53 regulated miR, which is normally downregulated in colon cancer, was upregulated by 2-fold (along with p21waf1/cip1) following IGF-1R inhibition only in p53 wild type cells. More importantly, we found that expression of miR-215 was decreased by 10-12 folds in chemosurviving or CD44+ fraction compared to parental or CD44- fraction of HCT-116 cells respectively. On the other hand, expression of thymidylate synthase (TS), the putative target of miR-215, overexpression of which is associated with 5-FU resistance in colon cancer was increased by 3- 10 fold in CD44+ and chemosurviving HCT-116 cells. Consistent with the above observation, treatment of colon cancer cells with 5-FU in combination with IGF-1R depletion resulted in robust 75% inhibition of colonosphere formation which was significantly higher than either modality alone only in p53 wild type HCT-116 cells. Conclusions: Depletion of IGF-1R resulted in reduction of CSC phenotype in colorectal cancer cells, which could partly be due to induction of p53 dependent miR-215 and down regulation of TS, its putative target. No significant financial relationships to disclose.
Chronic renal failure (CRF) was produced in female Sprague-Dawley rats by 7/8 nephrectomy. Creatinine clearance was depressed significantly (P less than 0.005) and blood urea nitrogen (BUN) increased in CRF rats when compared with the sham-operated (S) controls. CRF caused no apparent change in body weight but significantly increased pancreatic weight as well as increased DNA, RNA, and protein content. Pancreatic protein-to-DNA and RNA-to-DNA ratios were also found to be significantly higher in CRF rats than in the S controls. Trypsin-like activity and immunoreactive cationic trypsinogen levels were both increased in the pancreas of CRF rats, but not in their serum. On the other hand, protease inhibitory activity in the pancreas and serum was significantly decreased by CRF. The ability of the dispersed pancreatic acini isolated from CRF rats to incorporate [3H]-leucine into protein, in the absence and presence of 0.25 nM cholecystokinin octapeptide (CCK-8), was found to be lower than in the controls. Furthermore, discharge of both trypsinogen and chymotrypsinogen induced by CCK-8 was markedly reduced from acini of CRF rats as compared with the S controls. In contrast, lactate dehydrogenase (LDH) was released more readily from pancreatic acini of CRF. It is concluded that mild CRF produces hyperplasia and hypertrophy of the pancreas and lowers the responsiveness of acini to CCK-8 with respect to synthesis and secretion of proteins.
Groups of rats were injected with either saline or pentagastrin (0.5, 1 or 2 mg/kg/day) for 14 days. Pancreatic weight, DNA, RNA and protein contents as well as the ability of the pancreatic polyribosomes to synthesize protein in a cell-free system were investigated. In all three pentagastrin-treated groups the weight of the pancreas was found to be significantly higher (33–73%) than in the control. DNA, RNA and protein contents were significantly increased by 47, 98 and 85%, respectively, in the group that received the highest dose of the hormone. With respect to ribosomal protein synthesis, while the pentagastrin dose of 1 and 2 mg/kg/day resulted in 25 and 72% augmentation, respectively, the dose of 0.5 mg/kg had no influence on protein synthesis. It is concluded that chronic administration of pentagastrin (2 mg/kg/day for 14 days) causes hyperplasia and hypertrophy of the pancreas.
The effect of bilateral adrenalectomy and subsequent force-feeding of L-tryptophan on the gastric mucosal pepsin activity and [3H]leucine incorporation into total protein of the stomach (fundus) in vivo were investigated. One month after bilateral adrenalectomy the gastric mucosal pepsin activity and overall protein synthesis in the stomach were decreased by 72% and 52%, respectively. Twenty-four hours after a single tube-feeding of tryptophan (30 mg/100 g body weight) both activities returned to sham-operated control levels. In adrenalectomized rats the tryptophan-mediated stimulation of gastric mucosal pepsin activity was found to be sensitive to the RNA synthesis inhibitor, actinomycin-D. The diminution in gastric mucosal pepsin activity after adrenalectomy and its enhancement by tryptophan could not be related to the presence of an inhibitor or activator in the tissue. One month after adrenalectomy serum gastrin concentration was found to be 36% above that of the sham-operated control. In adrenalectomized rats, 24 and 48 h after tryptophan force-feeding, serum gastrin concentrations were decreased by 50% and 20%, respectively, but none of the values differed significantly from those of water-fed adrenalectomized controls.
The current study was undertaken to investigate the effect of differentially formulated polyphenolic compound Essential Turmeric Oil-Curcumin (ETO-Cur), and Tocotrienol-rich fraction (TRF) of vitamin E isomers on colorectal cancer (CRC) cells that produce aggressive tumors. Combinations of ETO-Cur and TRF were used to determine the combinatorial effects of ETO-Cur and TRF-mediated inhibition of growth of CRC cells in vitro and HCT-116 cells xenograft in SCID mice. 16S rRNA gene sequence profiling was performed to determine the outcome of gut microbial communities in mice feces between control and ETO-Cur-TRF groups. Bacterial identifications were validated by performing SYBR-based Real Time (RT) PCR. For metagenomics analysis to characterize the microbial communities, multiple software/tools were used, including Quantitative Insights into Microbial Ecology (QIIME) processing tool. We found ETO-Cur and TRF to synergize and that the combination of ETO-Cur-TRF significantly inhibited growth of HCT-116 xenografts in SCID mice. This was associated with a marked alteration in microbial communities and increased microbial OTU (operation taxonomic unit) number. The relative abundance of taxa was increased and the level of microbial diversity after 34 days of combinatorial treatment was found to be 44% higher over the control. Shifting of microbial family composition was observed in ETO-Cur-TRF treated mice as evidenced by marked reductions in Bacteroidaceae, Ruminococcaceae, Clostridiales, Firmicutes and Parabacteroids families, compared to controls. Interestingly, during the inhibition of tumor growth in ETO-Cur treated mice, probiotic Lactobacillaceae and Bifidobacteriaceae were increased by 20-fold and 6-fold, respectively. The relative abundance of anti-inflammatory Clostridium XIVa was also increased in ETO-Cur-TRF treated mice when compared with the control. Our data suggest that ETO-Cur-TRF show synergistic effects in inhibiting colorectal cancer cell proliferation in vitro and in mouse xenografts in vivo, and might induce changes in microbial diversity in mice.
Increasing evidence supports the contention that many malignancies, including sporadic colorectal cancer, are driven by the self-renewing, chemotherapy-resistant cancer stem/stem-like cells (CSC/CSLC), underscoring the need for improved preventive and therapeutic strategies targeting CSCs/CSLCs. Omega-3 polyunsaturated fatty acids (ω-3 PUFA), have been reported to inhibit the growth of primary tumors, but their potential as a preventive agent for recurring cancers is unexplored. The primary objectives of this investigation are (i) to examine whether eicosapentaenoic acid (EPA; one of the ω-3 PUFA) synergizes with FuOx (5-FU+Oxaliplatin), the backbone of colon cancer chemotherapy, and (ii) whether EPA by itself or in combination with conventional chemotherapy prevents the recurrence of colon cancer via eliminating/suppressing CSCs/CSLCs. FuOx-resistant (chemoresistant; CR) colon cancer cells, highly enriched in CSCs, were used for this study. Although EPA alone was effective, combination of EPA and FuOx was more potent in (i) inhibiting cell growth, colonosphere formation, and sphere-forming frequency, (ii) increasing sphere disintegration, (iii) suppressing the growth of SCID mice xenografts of CR colon cancer cells, and (iv) decreasing proinflammatory metabolites in mice. In addition, EPA + FuOx caused a reduction in CSC/CSLC population. The growth reduction by this regimen is the result of increased apoptosis as evidenced by PARP cleavage. Furthermore, increased pPTEN, decreased pAkt, normalization of β-catenin expression, localization, and transcriptional activity by EPA suggests a role for the PTEN-Akt axis and Wnt signaling in regulating this process. Our data suggest that EPA by itself or in combination with FuOx could be an effective preventive strategy for recurring colorectal cancer.
