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Wafik S Eldeiry - One of the best experts on this subject based on the ideXlab platform.
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small molecule Prodigiosin restores p53 tumor suppressor activity in chemoresistant colorectal cancer stem cells via c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2016Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract: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.
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abstract 1215 small molecule Prodigiosin mediated p53 pathway restoration and inhibition of self renewal in colorectal cancer involves c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2015Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract:Tumor suppressor p53 is frequently mutated or inactivated in colorectal cancer while p53 family member p73 is rarely mutated in cancer cells. Small molecules that activate p73 can elicit a p53-like tumor suppressive function and represent a novel approach for p53 pathway restoration. Colorectal tumors contain a small population of cancer stem cells (CSCs) capable of self-renewal that contributes to tumor maintenance and resistance to therapy. Targeting CSCs could improve treatment response and prolong patient survival. We have previously shown that small molecule Prodigiosin restores the p53 pathway via activation of p73 and inhibition of the mutant p53-p73 interaction (Hong et al., Cancer Research 74:1153-1165, 2014). We now demonstrate that Prodigiosin targets 5-Fluorouracil-resistant CSCs along with bulk tumor cells. Prodigiosin prevents colonosphere formation of CRC cell lines independent of p53 status of the cells. Prodigiosin reduces the viability of both CSCs and non-CSCs while 5-Fluorouracil only targets non-CSCs. Prodigiosin significantly reduces the growth of xenograft tumors initiated in mice with CSCs without toxic effects and prevents the passage of these tumors. In a p53-reponsive luciferase reporter assay, Prodigiosin induces p53-pathway transcription in colonospheres and CSC-initiated xenograft tumors. Stable shRNA knockdown of p73 revealed that Prodigiosin inhibits the self-renewal of CSCs in a p73-dependent manner. Next, we explored the mechanisms of Prodigiosin-mediated p53-pathway restoration and anti-CSC effects upstream of p73 activation. The oncogenic N-terminally truncated isoform ΔNp73 is a dominant negative inhibitor of p73 and p53. ΔNp73 levels correlate with poor overall survival in colorectal cancer patients. Western blot analysis revealed that Prodigiosin increases protein levels of p73 and its target genes and reduces levels of the oncogenic isoform ΔNp73. Prodigiosin has been previously shown to increase the expression of c-Jun, a member of the AP-1 family of transcription factors. c-Jun is known to regulate the induction of p73 and degradation of ΔNp73 in response to cellular stress. We hypothesized that Prodigiosin mediated p53-pathway restoration involves c-Jun upregulation resulting in ΔNp73 inhibition and p73 activation. Western blot analysis revealed that Prodigiosin induces levels of c-Jun and phospho-c-Jun. siRNA knockdown of c-Jun protein levels reduced Prodigiosin-mediated ΔNp73 downregulation. Thus, we have characterized a previously unrecognized mechanism of Prodigiosin-mediated p73 activation via c-Jun-dependent ΔNp73 inhibition. Ongoing experiments involve further validation of the mechanism by studying the effects of c-Jun knockdown and ΔNp73 overexpression on Prodigiosin-mediated effects on apoptosis, CSC self-renewal and p53 pathway restoration. Citation Format: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E. Allen, Amriti Lulla, David T. Dicker, Wafik S. El-Deiry. Small molecule Prodigiosin-mediated p53 pathway restoration and inhibition of self-renewal in colorectal cancer involves c-Jun-mediated ΔNp73 inhibition and p73 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1215. doi:10.1158/1538-7445.AM2015-1215
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Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53
Cancer Research, 2014Co-Authors: Bo Hong, Varun Vijay Prabhu, Shengliang Zhang, David T Dicker, Pieter A J Van Den Heuvel, Levy Kopelovich, Wafik S EldeiryAbstract:p53 reactivation offers a broad-based strategy for cancer therapy. In this study, we report the identification of Prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53-deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell-cycle arrest and apoptosis in p53-deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot TP53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting antitumor effects. The disruption of mutant p53/p73 interaction was specific to Prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, Prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by upregulating p73 and targeting mutant p53/p73 interaction there.
Chia Che Chang - One of the best experts on this subject based on the ideXlab platform.
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enhancing production of Prodigiosin from serratia marcescens c3 by statistical experimental design and porous carrier addition strategy
Biochemical Engineering Journal, 2013Co-Authors: Jo Shu Chang, Chia Che Chang, Wei Chuan Chen, Shih Horng Huang, Chih Hung Chang, Shanyu ChenAbstract:Abstract Serratia marcescens C3 produces a natural red-pigment, Prodigiosin, which exhibits immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. This work seeks to improve the production of Prodigiosin by S. marcescens C3 using various strategies. Starch and peptone were identified as the optimized carbon and nitrogen sources for the production of Prodigiosin, yielding a Prodigiosin concentration of 2.3 g/L. This value was significantly increased to 6.7 g/L using a carbon/nitrogen ratio of 6/4 (starch/peptone = 16 g/L/10.67 g/L). To enhance Prodigiosin production even further, a statistical experimental design methodology was utilized to optimize the composition of the culture medium that is utilized in the production of Prodigiosin. Prodigiosin production of 7.07 g/L was achieved when the concentrations of two trace compounds, FeSO 4 ·4H 2 O and MnSO 4 ·4H 2 O, were optimized using the statistical experimental design methodology. Their optimal concentrations were 0.56 mM and 3.25 mM, respectively. Ultimately, the production of Prodigiosin was increased from 2.3 g/L to 15.6 g/L, or by a factor of nearly seven by immobilizing microorganisms in 3% calcium alginate beads.
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Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines
Toxicology and applied pharmacology, 2012Co-Authors: Mu-yun Pan, Yu Ta Peng, Yuh-chiang Shen, Shu-yi Yang, Chia Che ChangAbstract:Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in Prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, Prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming Prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered Prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in Prodigiosin-induced cytotoxic ER stress response. In addition, Prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked Prodigiosin-induced PARP cleavage and greatly enhanced the survival of Prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates Prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired Prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of Prodigiosin's tumoricidal effect. Mechanistically, Prodigiosin engages the IRE1-JNK and PERK-eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death.
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Prodigiosin down-regulates SKP2 to induce p27KIP1 stabilization and antiproliferation in human lung adenocarcinoma cells
British journal of pharmacology, 2012Co-Authors: Hsin Ying Hsieh, Shin Chang Lin, Jo Shu Chang, Mu-yun Pan, Shu-yi Yang, Jeng-jer Shieh, Chun Jung Chen, Alan Yueh Luen Lee, Chia Che ChangAbstract:BACKGROUND AND PURPOSE High levels of SKP2 are a poor prognostic factor in multiple human cancers and mostly correlate with low p27KIP1 levels. Prodigiosin is a bacterial tripyrrole pigment with strong pro-apoptotic activity. Induction of cell cycle blockade underlies one of its anticancer actions but the mechanisms involved are unclear. The aim of this study was to explore the role of the SKP2–p27KIP1 axis in Prodigiosin's cytostatic effect on human lung adenocarcinoma cells. EXPERIMENTAL APPROACH Prodigiosin's effects on cell cycle progression and long-term cell proliferation of human lung adenocarcinoma cells were characterized by flow cytometry and colony formation assay, respectively. Real-time RT-PCR and promoter activity analyses were performed for assessing transcriptional control, while cycloheximide chase analysis evaluated protein stability. Immunoblotting was employed for mechanistic study. KEY RESULTS Prodigiosin increased p27KIP1 expression mainly by stabilizing p27KIP1 through transcriptional repression of SKP2. Importantly, SKP2 overexpression or p27KIP1 depletion restored the colony forming capacity of Prodigiosin-treated cells. Furthermore, Prodigiosin induced PKB dephosphorylation, leading to PKB inhibition as revealed by decreased serine 9 phosphorylation of GSK-3β. Constitutive PKB activation reduced Prodigiosin-induced SKP2 repression. Prodigiosin also down-regulated E2F1 (mediates PI3K/PKB-induced SKP2 transcription), but E2F1 overexpression failed to restore SKP2 expression in Prodigiosin-treated cells. CONCLUSIONS AND IMPLICATIONS Transcriptional repression of SKP2 and the consequent accumulation of p27KIP1 are essential for Prodigiosin's antiproliferative action. Mechanistically, Prodigiosin induces PKB inhibition to down-regulate SKP2 in a GSK-3β- and E2F1-independent manner. Our findings further implicate the potential for developing Prodigiosin as a novel class of SKP2-targeting anticancer agent.
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Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines
Toxicology and applied pharmacology, 2008Co-Authors: Yu Ta Peng, Show-mei Chuang, Shin Chang Lin, Bo Lin Feng, Jo Shu Chang, Chia Che ChangAbstract:Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby Prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in Prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that Prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as Prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to Prodigiosin, indicating an essential role of survivin down-regulation in Prodigiosin-induced apoptosis. In addition, we found that Prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of Prodigiosin is ascribed to the lowering of survivin expression, because Prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating Prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of Prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.
Varun Vijay Prabhu - One of the best experts on this subject based on the ideXlab platform.
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small molecule Prodigiosin restores p53 tumor suppressor activity in chemoresistant colorectal cancer stem cells via c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2016Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract: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.
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abstract 1215 small molecule Prodigiosin mediated p53 pathway restoration and inhibition of self renewal in colorectal cancer involves c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2015Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract:Tumor suppressor p53 is frequently mutated or inactivated in colorectal cancer while p53 family member p73 is rarely mutated in cancer cells. Small molecules that activate p73 can elicit a p53-like tumor suppressive function and represent a novel approach for p53 pathway restoration. Colorectal tumors contain a small population of cancer stem cells (CSCs) capable of self-renewal that contributes to tumor maintenance and resistance to therapy. Targeting CSCs could improve treatment response and prolong patient survival. We have previously shown that small molecule Prodigiosin restores the p53 pathway via activation of p73 and inhibition of the mutant p53-p73 interaction (Hong et al., Cancer Research 74:1153-1165, 2014). We now demonstrate that Prodigiosin targets 5-Fluorouracil-resistant CSCs along with bulk tumor cells. Prodigiosin prevents colonosphere formation of CRC cell lines independent of p53 status of the cells. Prodigiosin reduces the viability of both CSCs and non-CSCs while 5-Fluorouracil only targets non-CSCs. Prodigiosin significantly reduces the growth of xenograft tumors initiated in mice with CSCs without toxic effects and prevents the passage of these tumors. In a p53-reponsive luciferase reporter assay, Prodigiosin induces p53-pathway transcription in colonospheres and CSC-initiated xenograft tumors. Stable shRNA knockdown of p73 revealed that Prodigiosin inhibits the self-renewal of CSCs in a p73-dependent manner. Next, we explored the mechanisms of Prodigiosin-mediated p53-pathway restoration and anti-CSC effects upstream of p73 activation. The oncogenic N-terminally truncated isoform ΔNp73 is a dominant negative inhibitor of p73 and p53. ΔNp73 levels correlate with poor overall survival in colorectal cancer patients. Western blot analysis revealed that Prodigiosin increases protein levels of p73 and its target genes and reduces levels of the oncogenic isoform ΔNp73. Prodigiosin has been previously shown to increase the expression of c-Jun, a member of the AP-1 family of transcription factors. c-Jun is known to regulate the induction of p73 and degradation of ΔNp73 in response to cellular stress. We hypothesized that Prodigiosin mediated p53-pathway restoration involves c-Jun upregulation resulting in ΔNp73 inhibition and p73 activation. Western blot analysis revealed that Prodigiosin induces levels of c-Jun and phospho-c-Jun. siRNA knockdown of c-Jun protein levels reduced Prodigiosin-mediated ΔNp73 downregulation. Thus, we have characterized a previously unrecognized mechanism of Prodigiosin-mediated p73 activation via c-Jun-dependent ΔNp73 inhibition. Ongoing experiments involve further validation of the mechanism by studying the effects of c-Jun knockdown and ΔNp73 overexpression on Prodigiosin-mediated effects on apoptosis, CSC self-renewal and p53 pathway restoration. Citation Format: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E. Allen, Amriti Lulla, David T. Dicker, Wafik S. El-Deiry. Small molecule Prodigiosin-mediated p53 pathway restoration and inhibition of self-renewal in colorectal cancer involves c-Jun-mediated ΔNp73 inhibition and p73 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1215. doi:10.1158/1538-7445.AM2015-1215
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Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53
Cancer Research, 2014Co-Authors: Bo Hong, Varun Vijay Prabhu, Shengliang Zhang, David T Dicker, Pieter A J Van Den Heuvel, Levy Kopelovich, Wafik S EldeiryAbstract:p53 reactivation offers a broad-based strategy for cancer therapy. In this study, we report the identification of Prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53-deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell-cycle arrest and apoptosis in p53-deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot TP53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting antitumor effects. The disruption of mutant p53/p73 interaction was specific to Prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, Prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by upregulating p73 and targeting mutant p53/p73 interaction there.
Robert M. Q. Shanks - One of the best experts on this subject based on the ideXlab platform.
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Thermoregulation of Prodigiosin Biosynthesis by Serratia marcescens is Controlled at the Transcriptional Level and Requires HexS.
Polish journal of microbiology, 2019Co-Authors: Eric G. Romanowski, Nicholas A. Stella, Kara M. Lehner, Natalie C. Martin, Kriya R. Patel, Jake D. Callaghan, Robert M. Q. ShanksAbstract:Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite Prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the Prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the Prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow Prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of Prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of Prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level. Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite Prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the Prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the Prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow Prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of Prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of Prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.
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The LysR Transcription Factor, HexS, Is Required for Glucose Inhibition of Prodigiosin Production by Serratia marcescens.
Advances in microbiology, 2012Co-Authors: Nicholas A. Stella, James E. Fender, Roni M. Lahr, Eric J. Kalivoda, Robert M. Q. ShanksAbstract:Generation of many useful microbe-derived secondary metabolites, including the red pigment Prodigiosin of the bacterium Serratia marcescens, is inhibited by glucose. In a previous report, a genetic approach was used to determine that glucose dehydrogenase activity (GDH) is required for inhibiting Prodigiosin production and transcription of the Prodigiosin biosynthetic operon (pigA-N). However, the transcription factor(s) that regulate this process were not characterized. Here we tested the hypothesis that HexS, a LysR-family transcription factor similar to LrhA of Escherichia coli, is required for inhibition of Prodigiosin by growth in glucose. We observed that mutation of the hexS gene in S. marcescens allowed the precocious production of Prodigiosin in glucose-rich medium conditions that completely inhibited Prodigiosin production by the wild type. Unlike previously described mutants able to generate Prodigiosin in glucose-rich medium, hexS mutants exhibited GDH activity and medium acidification similar to the wild type. Glucose inhibittion of pigA expression was shown to be dependent upon HexS, suggesting that HexS is a key transcription factor in secondary metabolite regulation in response to medium pH. These data give insight into the Prodigiosin regulatory pathway and could be used to enhance the production of secondary metabolites.
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serratia marcescens quinoprotein glucose dehydrogenase activity mediates medium acidification and inhibition of Prodigiosin production by glucose
Applied and Environmental Microbiology, 2012Co-Authors: James E. Fender, Nicholas A. Stella, Roni M. Lahr, Eric J. Kalivoda, Cody M Bender, Robert M. Q. ShanksAbstract:ABSTRACT Serratia marcescens is a model organism for the study of secondary metabolites. The biologically active pigment Prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine), like many other secondary metabolites, is inhibited by growth in glucose-rich medium. Whereas previous studies indicated that this inhibitory effect was pH dependent and did not require cyclic AMP (cAMP), there is no information on the genes involved in mediating this phenomenon. Here we used transposon mutagenesis to identify genes involved in the inhibition of Prodigiosin by glucose. Multiple genetic loci involved in quinoprotein glucose dehydrogenase (GDH) activity were found to be required for glucose inhibition of Prodigiosin production, including pyrroloquinoline quinone and ubiquinone biosynthetic genes. Upon assessing whether the enzymatic products of GDH activity were involved in the inhibitory effect, we observed that d-glucono-1,5-lactone and d-gluconic acid, but not d-gluconate, were able to inhibit Prodigiosin production. These data support a model in which the oxidation of d-glucose by quinoprotein GDH initiates a reduction in pH that inhibits Prodigiosin production through transcriptional control of the Prodigiosin biosynthetic operon, providing new insight into the genetic pathways that control Prodigiosin production. Strains generated in this report may be useful in large-scale production of secondary metabolites.
Bo Hong - One of the best experts on this subject based on the ideXlab platform.
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small molecule Prodigiosin restores p53 tumor suppressor activity in chemoresistant colorectal cancer stem cells via c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2016Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract: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.
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abstract 1215 small molecule Prodigiosin mediated p53 pathway restoration and inhibition of self renewal in colorectal cancer involves c jun mediated δnp73 inhibition and p73 activation
Cancer Research, 2015Co-Authors: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E Allen, Amriti R Lulla, David T Dicker, Wafik S EldeiryAbstract:Tumor suppressor p53 is frequently mutated or inactivated in colorectal cancer while p53 family member p73 is rarely mutated in cancer cells. Small molecules that activate p73 can elicit a p53-like tumor suppressive function and represent a novel approach for p53 pathway restoration. Colorectal tumors contain a small population of cancer stem cells (CSCs) capable of self-renewal that contributes to tumor maintenance and resistance to therapy. Targeting CSCs could improve treatment response and prolong patient survival. We have previously shown that small molecule Prodigiosin restores the p53 pathway via activation of p73 and inhibition of the mutant p53-p73 interaction (Hong et al., Cancer Research 74:1153-1165, 2014). We now demonstrate that Prodigiosin targets 5-Fluorouracil-resistant CSCs along with bulk tumor cells. Prodigiosin prevents colonosphere formation of CRC cell lines independent of p53 status of the cells. Prodigiosin reduces the viability of both CSCs and non-CSCs while 5-Fluorouracil only targets non-CSCs. Prodigiosin significantly reduces the growth of xenograft tumors initiated in mice with CSCs without toxic effects and prevents the passage of these tumors. In a p53-reponsive luciferase reporter assay, Prodigiosin induces p53-pathway transcription in colonospheres and CSC-initiated xenograft tumors. Stable shRNA knockdown of p73 revealed that Prodigiosin inhibits the self-renewal of CSCs in a p73-dependent manner. Next, we explored the mechanisms of Prodigiosin-mediated p53-pathway restoration and anti-CSC effects upstream of p73 activation. The oncogenic N-terminally truncated isoform ΔNp73 is a dominant negative inhibitor of p73 and p53. ΔNp73 levels correlate with poor overall survival in colorectal cancer patients. Western blot analysis revealed that Prodigiosin increases protein levels of p73 and its target genes and reduces levels of the oncogenic isoform ΔNp73. Prodigiosin has been previously shown to increase the expression of c-Jun, a member of the AP-1 family of transcription factors. c-Jun is known to regulate the induction of p73 and degradation of ΔNp73 in response to cellular stress. We hypothesized that Prodigiosin mediated p53-pathway restoration involves c-Jun upregulation resulting in ΔNp73 inhibition and p73 activation. Western blot analysis revealed that Prodigiosin induces levels of c-Jun and phospho-c-Jun. siRNA knockdown of c-Jun protein levels reduced Prodigiosin-mediated ΔNp73 downregulation. Thus, we have characterized a previously unrecognized mechanism of Prodigiosin-mediated p73 activation via c-Jun-dependent ΔNp73 inhibition. Ongoing experiments involve further validation of the mechanism by studying the effects of c-Jun knockdown and ΔNp73 overexpression on Prodigiosin-mediated effects on apoptosis, CSC self-renewal and p53 pathway restoration. Citation Format: Varun Vijay Prabhu, Shengliang Zhang, Bo Hong, Joshua E. Allen, Amriti Lulla, David T. Dicker, Wafik S. El-Deiry. Small molecule Prodigiosin-mediated p53 pathway restoration and inhibition of self-renewal in colorectal cancer involves c-Jun-mediated ΔNp73 inhibition and p73 activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1215. doi:10.1158/1538-7445.AM2015-1215
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Prodigiosin rescues deficient p53 signaling and antitumor effects via upregulating p73 and disrupting its interaction with mutant p53
Cancer Research, 2014Co-Authors: Bo Hong, Varun Vijay Prabhu, Shengliang Zhang, David T Dicker, Pieter A J Van Den Heuvel, Levy Kopelovich, Wafik S EldeiryAbstract:p53 reactivation offers a broad-based strategy for cancer therapy. In this study, we report the identification of Prodigiosin that can reactivate p53 family-dependent transcriptional activity in p53-deficient human colon cancer cells. Prodigiosin and its structural analogue (compound R) induced the expression of p53 target genes accompanied by cell-cycle arrest and apoptosis in p53-deficient cancer cells. Prodigiosin restored p53 signaling in cancer cells harboring hotspot TP53 mutations, with little to no detectable cytotoxicity in normal human fibroblasts and with no genotoxicity. Prodigiosin induced the expression of p73 and disrupted its interaction with mutant p53, thereby rescuing p53 pathway deficiency and promoting antitumor effects. The disruption of mutant p53/p73 interaction was specific to Prodigiosin and not related to mTOR inhibition. Our findings suggest that mutant p53 needs to be targeted in the context of p73 stimulation to allow efficient restoration of the p53 pathway. In exhibiting this capability, Prodigiosin and its analogue provide lead compounds to rescue deficiencies in the p53 pathway in cancer cells by upregulating p73 and targeting mutant p53/p73 interaction there.