Belinostat, at Its Clinically Relevant Concentrations, Inhibits Rifampicin-Induced CYP3A4 and MDR1 Gene Expression

Abstract

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...

Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity

“…This work further confirms the upregulated metabolic expression of CYP3A4 decreases doxorubicin efficacy in confirmation with previous studies across multiple cancer types, including liver ( 58 ), colorectal ( 29 ), breast ( 59 ), and prostate ( 60 ). Inhibition of CYP3A4 activity of human primary hepatocytes has shown to suppress human pregnane X receptor (hPXR) agonist-induced chemoresistance ( 61 ). Overexpression of CYP3A4 in tumor tissues and chemoresistance to therapeutics has been shown in clinical practices ( 62 ).…”

Gefitinib Inhibits Rifampicin-Induced CYP3A4 Gene Expression in Human Hepatocytes

“…It is interesting to compare GEF binding to hPXR with that of our previously published antagonist, belinostat. 13 Primarily, GEF with a rigid bicyclic ring is constrained in its conformational space ( Figure 1 ). On the other hand, belinostat with monocyclic rings is more flexible and has the capacity to form strong hydrogen bonds with the terminal N=O group and with the SO 2 group on the other end of the molecule, which is easily accessible to the polar/charged groups of PXR groups ( Figure 5 in ( 13 )).…”

“… 13 Primarily, GEF with a rigid bicyclic ring is constrained in its conformational space ( Figure 1 ). On the other hand, belinostat with monocyclic rings is more flexible and has the capacity to form strong hydrogen bonds with the terminal N=O group and with the SO 2 group on the other end of the molecule, which is easily accessible to the polar/charged groups of PXR groups ( Figure 5 in ( 13 )). Thus, belinostat interaction at the AF2 site is more dominated by hydrogen bonds and that of GEF is dominated by hydrophobic interactions, resulting in a slightly higher docking score of belinostat.…”

Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity

“…This work further confirms the upregulated metabolic expression of CYP3A4 decreases doxorubicin efficacy in confirmation with previous studies across multiple cancer types, including liver 58 , colorectal 29 , breast 59 , and prostate 60 . Inhibition of CYP3A4 activity of human primary hepatocytes has shown to suppress human pregnane X receptor (hPXR) agonist-induced chemoresistance 61 . Overexpression of CYP3A4 in tumor tissues and chemoresistance to therapeutics has been shown in clinical practices 62 .…”