Cobalamin Potentiates Vinblastine Cytotoxicity Through Downregulation of mdr-1 Gene Expression in HepG2 Cells

Abstract

Background: P-glycoprotein (Pgp), produced by multidrug resistance-1 gene (mdr-1), is a main mechanism developed by cancer cells to guard against anti-cancer drugs. Alterations of DNA methylation of the mdr-1 gene promoter are known to be linked to mdr-1 gene expression and are probably related to intracellular S-adenosyl-methionine. We here used HepG2 cells to determine the role of the methionine cycle (through the use of the Methionine-Synthase (MS) cofactor, cobalamin) on mdr-1 gene expression. Methods: Semiquantitative RT-PCR of mdr-1 gene, cellular retention of rhodamine-123, and vinblastine cytotoxicity were carried out on cells cultivated with and without cobalamin. Methylation status of the mdr-1 gene promoter was determined by methylation-specific PCR. Results: Addition of cobalamin to the cells led to an increase in MS activity, to a significant decrease in mdr-1 gene expression which is correlated to an increase in retention of the Pgp substrate Rhodamine 123. Furthermore, cobalamin potentiated cell sensitivity to vinblastine to the same range as that of the Pgp blocker verapamil and prevented methotrexate-induced up-regulation of mdr-1 gene expression. However, no modification in methylation of the mdr-1 gene promoter was observed. Conclusion: Cobalamin downregulates mdr-1 gene expression, as well as Pgp expression and function, and significantly increases cytotoxicity of vinblastine. The identification of this novel way of diminishing cellular resistance to the chemotherapeutic agent vinblastine holds promises of leading to better treatments for cancer patients.

Cobalamin and normal prions: A new horizon for cobalamin neurotrophism

“…However, there are many arguments against the hypothesis that the increase is merely due to the selective DNA hypomethylation of PrP C gene(s): (i) the increase in SC PrP C levels is concomitant with a marked decrease in SC PrP C -mRNA levels [66]; (ii) it has been shown that Cbl induces the translational up-regulation of methionine synthase [87], a finding that is in some respects similar to our finding that Cbl regulates PrP C -mRNA levels in Cbl target tissues [66]; (iii) no changes in total protein content have been found in the SC of our Cbl-D rats (Riboni and Scalabrino, unpublished results); (iv) Cbl down-regulates the expression of multidrug resistance (mdr)-1 gene, but this effect is independent of the methylation status of the mdr-1 promoter [88]; (v) many vitamins regulate gene expression by means of different mechanisms not involving the degree of DNA methylation [89]; (vi) it has been demonstrated that Cbl-D neuropathy cannot reasonably be attributed to a failure of the methylation and/or synthesis of myelin basic protein in the rat CNS [90,91]; and (vii) rats made Cbl-D by means of total gastrectomy show increased TNF-a [92] and EGF [93] levels of livers 2 months after total gastrectomy, but these levels normalized 4 months after total gastrectomy, although other authors have demonstrated that the hepatic DNA of Cbl-D rats is hypomethylated [94]. The SC PrP C levels of TGX rats show the same trend [66].…”

Regulation of Biotransformation Systems and ABC Transporters by Benznidazole in HepG2 Cells: Involvement of Pregnane X-Receptor

“…The activity of P-gp in HepG2 cells was assessed measuring the intracellular content of the fluorescent compound Rh123, which inversely associates with the amount of substrate extruded [23], [29]. It is known that the probe is transported by P-gp and to some extent by BCRP [30].…”

Very important pharmacogene summary

“…Repressors of P-gp, including certain antineoplastic agents that act at nuclear receptors [73], or endotoxin [74], cobalamin [50], and atorvastatin [75,76], potentiate the action of substrates; whereas rifampin (rifampicin) [51] and cell stress signals induce P-gp-mediated drug resistance [29,50,75]. Another mechanism for P-gp-related pharmacoresistance to cytotoxic agents is hypothesized to relate to the cell stress signals they induce [52,77].…”

“…Upregulation of ABCB1 gene expression can occur at gene promoter sequences through transactivation [10,47,78], for example, by the pregnane X receptor ( NR1I2 ) gene in response to substrates that may have overlapping specificity for P-gp [29]; or induction can occur independent of nuclear receptors [79]. Alternatively, epigenetic inactivation of P-gp can occur by DNA methylation at specific nucleotide sequences within the promoter sequence, called CpG islands, as has been observed in some cancer tissues [80]; or downregulation of P-gp can also occur by mechanisms other than by DNA methylation, for example, in response to cobalamin, a vitamin B-12 derivative [50]. …”