prodigiosin and Colorectal-Neoplasms

Lactobacillus acidophilus ghosts (LAGs) with the unique safety of a probiotic, inherent tropism for colon cells, and multiple bioactivities offer promise as drug carriers for colon targeting. Our objective was to evaluate LAGs functionalized with prodigiosin (PG), apoptotic secondary bacterial metabolite, as a bioinspired formulation against colorectal cancer (CRC). LAGs were prepared by a chemical method and highly purified by density gradient centrifugation. LAGs were characterized by microscopic and staining techniques as relatively small-sized uniform vesicles (≈1.6 µm), nearly devoid of cytoplasmic and genetic materials and having a negatively charged intact envelope. PG was highly bound to LAGs envelope, generating a physiologically stable bioactive entity (PG-LAGs), as verified by multiple microscopic techniques and lack of PG release under physiological conditions. PG-LAGs were active against HCT116 CRC cells at both the cellular and molecular levels. Cell viability data highlighted the cytotoxicity of PG and LAGs and LAGs-induced enhancement of PG selectivity for HCT116 cells, anticipating dose reduction for PG and LAGs. Molecularly, expression of the apoptotic caspase 3 and P53 biomarkers in HCT116 intracellular proteins was significantly upregulated while that of the anti-apoptotic Bcl-2 (B-cell lymphoma 2) was downregulated by PG-LAGs relative to PG and 5-fluorouracil. PG-LAGs provide a novel bacteria-based combination for anticancer biomedicine.

Topics: Antineoplastic Agents; Apoptosis; Colorectal Neoplasms; Humans; Probiotics; Prodigiosin

Chemotherapy failure is a major cause of recurrence and poor prognosis in colorectal cancer (CRC) patients. Inhibition of autophagy is a promising strategy to augment the cytotoxicity of chemotherapeutic agents. We identified prodigiosin, a secondary metabolite produced by various bacteria, as a novel autophagy inhibitor that interfered with the autophagic flux in CRC cells by blocking autophagosome-lysosome fusion and lysosomal cathepsin maturation, resulting in the accumulation of LC3B-II and SQSTM. Suppression of autophagy by prodigiosin sensitized the CRC cells to 5-fluorouracil (5-Fu) in vitro, and the combination treatment markedly reduced cancer cell viability partly via caspase-dependent apoptosis. Furthermore, prodigiosin and 5-Fu synergistically inhibited CRC xenograft growth in vivo without any adverse effects. In conclusion, prodigiosin inhibits late stage autophagy and sensitizes tumor cells to 5-Fu, indicating its therapeutic potential in CRC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagosomes; Autophagy; Caco-2 Cells; Cell Death; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Fluorouracil; HCT116 Cells; HT29 Cells; Humans; Lysosomes; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Recurrence, Local; Prodigiosin

Tumor suppressor p53 is frequently mutated or inactivated in colorectal cancer. In contrast, p53 family member p73 is rarely mutated in colorectal cancer and p73 activation elicits p53-like tumor suppression. Colorectal cancer stem cells (CRCSC) comprise a rare self-renewing subpopulation that contributes to tumor maintenance and chemoresistance. p53 restoration is known to target CRCSCs, but p73 restoration in CRCSCs has not been examined. In this study, we investigated the effects of the small-molecule prodigiosin, which restores the p53 pathway in tumor cells via p73 activation, on CRCSCs in vitro and in vivo Prodigiosin prevented colonosphere formation independent of p53 status and reduced the viability of self-renewing, 5-fluorouracil-resistant Aldefluor positive [Aldefluor(+)] CRCSCs in vitro Furthermore, prodigiosin inhibited the growth of xenograft tumors initiated with Aldefluor+ cells without toxic effects and limited the tumorigenic potential of these cells. Consistently, prodigiosin induced activation of a p53-responsive luciferase reporter in colonospheres, Aldefluor(+) cells, and tumor xenografts. Mechanistic studies revealed that prodigiosin increased the levels of p73 and reduced levels of the oncogenic N-terminally truncated isoform ΔNp73 in Aldefluor(+) cells. Accordingly, p73 knockdown or ΔNp73 overexpression suppressed prodigiosin-mediated inhibition of colonosphere formation. Moreover, prodigiosin increased levels of the transcription factor c-Jun, a regulator of p73 and ΔNp73, in both the cytoplasm and nucleus. c-Jun knockdown attenuated prodigiosin-mediated p53-reporter activation, ΔNp73 downregulation, p73 activation, and cell death. Collectively, our findings highlight the previously uncharacterized use of p73-activating therapeutics to target CRCSCs. Cancer Res; 76(7); 1989-99. ©2016 AACR.

Topics: Animals; Anti-Bacterial Agents; Colorectal Neoplasms; Humans; Mice; Neoplastic Stem Cells; Nuclear Proteins; Prodigiosin; Signal Transduction; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential biomarkers could be targeted in CRC initiating cells with stem-like cells properties by prodigiosin and this compound with high pro-apoptotic capacity represents the possibility of its therapeutic application directed against CCSCs.

Topics: Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Down-Regulation; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; MicroRNAs; Neoplastic Stem Cells; Prodigiosin; Proto-Oncogene Mas; RNA, Messenger; Survivin

Colorectal cancer remains often refractory to classic therapies. In consequence, the search for new anti-tumor agents with minimal toxicity is of particular interest in colon cancer treatment. Prodigiosin as a secondary metabolite of Serratia marcescens induces apoptosis in various kinds of cancer cells with low toxicity on normal cells. In the present study, we evaluated the effect of prodigiosin on proliferation and expression of apoptotic-related genes in HT-29 cells. Malignant cells were treated to various concentrations of prodigiosin and proliferation rate, survivin, Bcl-2, Bax and Bad mRNA levels, caspase 3 activation and apoptosis were evaluated by different cellular and molecular techniques. Treatment of cells with increasing concentration of prodigiosin decreased significantly cell proliferation in a dose- and time-dependent manner. Following 48-h treatment, growth rate was measured to be 77 ± 6.8, 41.3 ± 3.1 and 46 ± 6.3 % for 100, 400 and 600 nM prodigiosin, respectively, compared to untreated cells. This molecule induced 61.7, 90 and 89 % decrease in survivin mRNA level as well as 1.9-, 2.8- and 2.2-fold increase in caspase 3 activation for indicated concentrations of prodigiosin, respectively. The level of Bcl-2 mRNA was inversely proportional to Bax and Bad mRNA levels. Low mRNA levels of Bcl-2 combined with high levels of Bax and Bad mRNAs were correlated to higher apoptosis rate in treated cells. Our data suggest that prodigiosin-induced apoptosis may ascribe to Bcl-2 and survivin inhibition in HT-29 cells and these genes may provide promising molecular targets of prodigiosin. Collectively, prodigiosin may have a great potential for colorectal cancer-directed therapy.

Topics: Adenocarcinoma; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Flow Cytometry; HT29 Cells; Humans; Polymerase Chain Reaction; Prodigiosin; Serratia marcescens