Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively.

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Activation of pregnane X receptor (PXR) by clinical compounds during multidrug chemotherapy results in upregulation of the expression of PXR target genes, including cytochrome p450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), leading to chemoresistance. It is possible to overcome the PXR‐mediated chemoresistance by downregulating the upregulated PXR target genes by inhibiting the activated PXR. However, a selective and less‐toxic PXR antagonist has yet to be developed. In this regard, a clinical anticancer drug, with selective PXR antagonistic activity at its less‐toxic concentrations, would be beneficial. We sought to determine whether belinostat, a clinically‐used histone deacetylase inhibitor, inhibits the PXR target gene expression at its clinically relevant plasma concentrations (< ~100 μM) in human hepatocytes (primary hepatocytes & hepatocells) and intestinal cells (LS174T colon cancer cells). Rifampicin, an agonist of human PXR, was used to activate PXR. Cell viability and CYQUANT cell proliferation assays were performed to determine cytotoxicity and cell proliferation, respectively. Quantitative RT‐PCR assays were conducted to study the gene expression. CYP3A4 p450‐Glo and Rhodamine‐123 intracellular accumulation assays were performed to determine the function of CYP3A4 and MDR1, respectively. Belinostat, at its unbound therapeutic plasma concentrations (< ~5 μM) did not affect the viability of LS174T cells and the hepatocytes. Belinostat (1 & 3 μM) not only inhibited rifampicin‐induced gene expression of CYP3A4 and MDR1, but also attenuated rifampicin‐induced activity of CYP3A4 and MDR1. However, belinostat alone did not affect CYP3A4 or MDR1 gene expression. These results suggest that belinostat does not affect the basal expression of PXR target genes but downregulates the upregulated PXR target genes by inhibiting the ligand‐activated PXR. Notably, belinostat, at its PXR inhibiting concentrations, decreased rifampicin‐induced proliferation of LS174T cells, suggesting that belinostat suppresses PXR‐mediated proliferation of the cancer cells. Interestingly, belinostat failed to inhibit rodent PXR agonist pregnenolone‐16 alpha‐carbonitrile (PCN)‐induced expression of CYP3A1 (the rat analog of human CYP3A4) in rat primary hepatocytes, suggesting that belinostat exhibits species‐specific inhibition of PXR at unbound plasma therapeutic concentrations. Taken together, these results are consistent with the conclusion that belinostat, at its less‐toxic and clinically relevant unbound plasma concentrations, inhibits the ligand‐activated human PXR target gene expression. Future studies will determine the mechanisms of belinostat inhibition of PXR, and belinostat sensitization of the cancer cells to chemotherapy drugs with PXR agonistic activity. Support or Funding Information The authors would like to thank Drs. Coleman, Schwartz, and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant...

Variations in the expression of human pregnane X receptor (hPXR)-mediated cytochrome p450 3A4 (CYP3A4) in liver can alter therapeutic response to a variety of drugs and may lead to potential adverse drug interactions. We sought to determine whether Mg 2+ /Mn 2+ -dependent phosphatase 1A (PPM1A) regulates hPXR-mediated CYP3A4 expression. PPM1A was found to be coimmunoprecipitated with hPXR. Genetic or pharmacologic activation of PPM1A led to a significant increase in hPXR transactivation of CYP3A4 promoter activity. In contrast, knockdown of endogenous PPM1A not only attenuated hPXR transactivation, but also increased proliferation of HepG2 human liver carcinoma cells, suggesting that PPM1A expression levels regulate hPXR, and that PPM1A expression is regulated in a proliferation-dependent manner. Indeed, PPM1A expression and hPXR transactivation were found to be significantly reduced in subconfluent HepG2 cells compared with confluent HepG2 cells, suggesting that both PPM1A expression and hPXR-mediated CYP3A4 expression may be downregulated in proliferating livers. Elevated PPM1A levels led to attenuation of hPXR inhibition by tumor necrosis factor-a and cyclin-dependent kinase-2, which are known to be upregulated and essential during liver regeneration. In mouse regenerating livers, similar to subconfluent HepG2 cells, expression of both PPM1A and the mouse PXR target gene cyp3a11 was found to be downregulated. Our results show that PPM1A can positively regulate PXR activity by counteracting PXR inhibitory signaling pathways that play a major role in liver regeneration. These results implicate a novel role for PPM1A in regulating hPXR-mediated CYP3A4 expression in hepatocytes and may explain a mechanism for CYP3A repression in regenerating livers.

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Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1”,2”:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1”,2”:1,5]pyrazolo[3,4-b]quinoline, exhibited more than tenfold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and submicromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and fivefold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer.

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