**6. Involvement of ABCC6/MRP6 in Drug Resistance**

It is well known that ABC transporters play an important role to maintain cellular physiology by transporting different substrates. On the other hand, despite the pathological involvement in certain crucial diseases, these transporters are also involved in drug resistance [39]. Similarly, ABCC family consisted of 13 members, which are involved in the transportation of different substrates. Nine of them are also implicated in the resistance to a variety of chemotherapeutic agents. Martin G. et al., in a study on Chinese hamster ovarian cancer cells (CHO), demonstrated that ABCC6 is not only able to mediate the efflux of glutathione conjugate but is also involved in resistance to some natural anti-cancer drugs, like doxorubicin, etoposide, actinomycin D, daunorubicin, and cisplatin [27]. Tyrosine Kinase Inhibitors (TKIs), like nilotinib and dasatinib, are effective treatments available for Chronic Myeloid Leukaemia (CML) and found to interact with ABCB1 and ABCG2. However, a recent study suggested that nilotinib and dasatinib might be substrates for ABCC6, whose over-expression is responsible for resistance of both TKIs [40].

To investigate the possible involvement of different transporters in drug resistance, we investigated the mRNA expression of ABCC6 and other ABC transporters mainly involved in drug resistance, such as ABCB1, ABCC1, and ABCG2, in the bone marrow samples obtained from acute myeloid leukemia (AML) patients. The samples obtained after diagnosis revealed no difference in the expression level of ABCC6 and ABCB1 between healthy control and AML patients. The expression of ABCC1 from AML patients was higher compared to the healthy individual. Moreover, the expression of ABCG2 was always found down-regulated in AML patients compared to controls. We also observed the age- and gender-associated changes in the expression level of the genes. Interestingly, ABCB1, ABCC1, and ABCC6 were less expressed in older patients compared to younger ones, whereas ABCC6 and ABCC1 were highly expressed in female patients. Additionally, only ABCG2 expression was found higher after chemotherapy, while no variation was found in ABCC1, ABCB1, and ABCC6. However, the expression of ABCC6 and ABCB1 was found to be up-regulated after the treatment with Trichostatin A (an inhibitor of histone deacetylase) and 5-Aza-2′deoxycytidine (an inhibitor of DNA methyltransferase) in AML cell line HL-60. These data reveal that changes in expression of ABCG2 before and after the treatment can be related to disease or as a therapeutic marker. On the other hand, the expression of ABCC6 and ABCB1 is transcriptionally or epigenetically controlled [41].

In addition, a study conducted by Jeon H-Metal [42] suggested that an inhibitor of differentiation 4 (ID4) increases the SOX2-mediated expression of ABCC6 and ABCC3 in glioma stem cells (GSC), which have the potential to initiate a brain tumor, show resistance to chemotherapy, and are responsible for higher recurrence rates of Glioblastoma multiforme (GBM) [43].

Drug resistance is the biggest problem for cancer therapy, for which many molecules have been synthesized and tested, but MDR in cancer therapy still persists. In order to identify the promising molecules to inhibit MRP6 and mitigate marginal drug resistance, we tested 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4] oxadiazolo [3,4-c][1,4] thiazin-3, also known as 2C and structurally similar to diltiazem. Surprisingly, the efflux of doxorubicin was reduced in 2C-treated cells. Moreover, cell esterase activity and H3 histone acetylation were reduced in 2C-treated cells, which suggests that 2C is not only able to mitigate drug resistance but also able to inhibit nucleophilic substitution reactions [44].

These findings confirm that ABCC6 is not only involved in the progression of the genetic disease PXE but is also involved in resistance to many anti-cancer agents [45,46].
