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Na(+)-coupled HCO(3)(-) transporters (NBCs) mediate the transport of bicarbonate ions across cell membranes and are thus ubiquitous regulators of intracellular pH. NBC dysregulation is associated with a range of diseases; for instance, NBCn1 is strongly up-regulated in a model of ErbB2-dependent breast cancer, a malignant and widespread cancer with no targeted treatment options, and single-nucleotide polymorphisms in NBCn1 genetically link to breast cancer development and hypertension. The N-cyanosulfonamide S0859 has been shown to selectively inhibit NBCs, and its availability on the gram scale is therefore of significant interest to the scientific community. Herein we describe a short and efficient synthesis of S0859 with an overall yield of 45 % from commercially available starting materials. The inhibitory effect of S0859 on recovery of intracellular pH after an acid load was verified in human and murine cancer cell lines in Ringer solutions. However, S0859 binds very strongly to components in plasma, and accordingly, measurements on isolated murine tissues showed no effect of S0859 at concentrations up to 50 μM.

Despite the frequent expression of N-terminally truncated ErbB2 (DNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how DNErbB2 affects chemotherapy-induced cell death. Previously it was shown that DNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na þ /H þ exchanger (SLC9A1/NHE1) strongly sensitizes DNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which DNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible DNErbB2 expression strongly reduced cisplatin-induced ATM-and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21 WAF1/Cip1 (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by DNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatininduced caspase-7 cleavage in DNErbB2-MCF-7 cells was Noxaand caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na þ /HCO 3 À cotransporter (SLC4A7/ NBCn1) was internalized following cisplatin exposure.Implications: This work reveals that DNErbB2 strongly affects several major pro-and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with DNErbB2 and/or NHE1 overexpression.

<p>Supplementary figure 3: Working model. Pathways involved in cisplatin-induced death in MCF-7 breast cancer cells, and their relation to ΔNErbB2 expression.</p>