Sequence-Dependent Diastereospecific and Diastereodivergent Crosslinking of DNA by Decarbamoylmitomycin C.

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug.

Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin†C.

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-ß-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-ß-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

Involvement of Akt in mitomycin C and its analog triggered cytotoxicity in MCF-7 and K562 cancer cells.

Mitomycin C (MC) is a well-known DNA alkylating agent. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger effects on cancer with p53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (p53-proficient) and K562 (p53-deficient) cells in a p53-independent mode. This study aimed to elucidate the upstream signaling pathway of p21WAF1/CIP1 activation triggered by MC/DMC. Besides p53, Akt plays an important role on deactivating p21WAF1/CIP1 . The results showed that MC/DMC inhibited Akt in MCF-7 cells, but not in K562 cells. By knocking down p53, the Akt inhibition in MCF-7 cells was alleviated. This implied that the deactivated Akt caused by MC/DMC was p53-dependent. With Akt activator (SC79), p21WAF1/CIP1 activation triggered by MC/DMC in MCF-7 cells was not reduced. This indicated that Akt inhibition triggered by MC/DMC was not associated with MC/DMC-induced p21WAF1/CIP1 activation. Label-free quantitative proteomic profiling analysis revealed that DMC has a stronger effect on down-regulating the PI3K/Akt signaling pathway in MCF-7 cells as compared to MC. No significant effect of MC/DMC on PI3K/Akt in K562 cells was observed. In summary, MC/DMC regulate Akt activation in a p53-dependent manner. This Akt deactivation is not associated with p21WAF1/CIP1 activation in response to MC/DMC.