Figure 6. Akt isoforms are concentration dependently reduced by YM-1, potential mechanism for resistance phenomena. MCF7 cells were treated with noted concentrations of YM-1 for 6 hours. Cells lysates were analyzed by Western blot (A). Densitometry analysis of Akt-1 and Akt2 levels shown as % of vehicle treatment 6 SD (B). doi:10.1371/journal.pone.0035566.g006 It is possible that the cytosolic presence of YM-1, versus mitochondrial of MKT-077, drives the increased toxicity and Akt clearance observe with YM-1. Though the mitochondrial aspects of MKT-077 are well characterized [1,2,3], recent data suggests that MKT-077 can also interact with cytosolic Hsp70 family members [22]. If MKT-077 is able to inhibit cytosolic Hsp70 as it does mortalin [4,5], YM-1 might also inhibit cytosolic Hsp70. References 1. Koya K, Li Y, Wang H, Ukai T, Tatsuta N, et al. (1996) MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation. Cancer Res 56: 538–543. 2. Britten CD, Rowinsky EK, Baker SD, Weiss GR, Smith L, et al. (2000) A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin Cancer Res 6: 42–49. 3. Propper DJ, Braybrooke JP, Taylor DJ, Lodi R, Styles P, et al. (1999) Phase I trial of the selective mitochondrial toxin MKT077 in chemo-resistant solid tumours. Ann Oncol 10: 923–927. 4. Wadhwa R, Sugihara T, Yoshida A, Nomura H, Reddel RR, et al. (2000) Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function. Cancer Res 60: 6818–6821. 5. Wadhwa R, Takano S, Robert M, Yoshida A, Nomura H, et al. (1998) Inactivation of tumor suppressor p53 by mot-2, a hsp70 family member. J Biol Chem 273: 29586–29591. Rhodacyanine Derivative Kills Resistant Cancers Hsp70 inhibitors have demonstrated cancer specificity as well as the ability to reduce Hsp70 client proteins [20,23,24,25]. We suspect that Hsp70 inhibition could be the mechanism of YM-1; however further examination is required. Tamoxifen therapies typically fail due to the development of resistance. Acquired resistances take time to develop. Our studies have demonstrated that brief treatments with YM-1 can re- sensitize refractory cancers to tamoxifen. The benefit of such a short treatment is the lack of opportunity for a resistance to YM-1 itself as well as reduced likelihood for off-target toxicities. Moreover, the ability to negate existing resistances allows for the reintroduction of putative chemotherapies; preventing the need for more costly and potentially dangerous secondary and tertiary therapeutic strategies. In addition, YM-1 treatment alone was able to selectively kill only certain cancer cells, suggesting not only tolerability to the approach but also a need for further characterization about the specific cell types that might be sensitive to these compounds and Akt depletion. These benefits to patients coupled with the high number of cancer varieties linked to Akt dysfunction provides a platform for the continued study and development of new compounds to deplete Akt through manipulation of the rhodocyanine scaffold. While other kinases have been identified to phosphorylated ERa, Akt is a major survival kinase, regardless of resistance phenotype, and its clearance could enhance the efficacy of chemotherapeutics. If our hypothesis that YM-1 is an Hsp70 inhibitor is accurate, this clearance could be due to enhanced ubiquitination of Akt, as the ubiquitin ligase for Akt, CHIP (carboxy terminus of Hsc70 interacting protein) [26], is known to interact with Hsp70 [27]. These studies demonstrate the need for more mechanistic insight into the mode of action of rhodacyanines. Minor changes between MKT-077 and YM-1 lead to an increased cytosolic presence that was able to maintain similar specificity. This suggests that modifications to this scaffold could elicit specific toxicity or reduce renal toxicity, as observed in the clinical and laboratory trials [1,2,3]. In fact, our data demonstrating an almost preferential killing of breast cancer cells, versus other cancer cell types, suggests that specificity for particular tumor varieties could be built into the rhodacyanine scaffold. Author Contributions Conceived and designed the experiments: CAD JEG J. Koren YM JQC. Performed the experiments: J. Koren YM J. Kiray JCO LN JG LJB XKL UKJ. Analyzed the data: J. Koren JCO CAD. Contributed reagents/ materials/analysis tools: JEG YM JQC JG. Wrote the paper: J. Koren CAD. 6. DeSantis C, Siegel R, Bandi P, Jemal A (2011) Breast cancer statistics, 2011. CA Cancer J Clin 61: 409–418. 7. Gonzalez-Angulo AM, Morales-Vasquez F, Hortobagyi GN (2007) Overview of resistance to systemic therapy in patients with breast cancer. Adv Exp Med Biol 608: 1–22. 8. Badia E, Oliva J, Balaguer P, Cavailles V (2007) Tamoxifen resistance and epigenetic modifications in breast cancer cell lines. Curr Med Chem 14: 3035–3045. 9. Cobleigh MA (2011) Other options in the treatment of advanced breast cancer. Semin Oncol 38 Suppl 2: S11–16. 10. Nahta R (2012) Pharmacological Strategies to Overcome HER2 Cross-Talk and Trastuzumab Resistance. Curr Med Chem. 11. Hudis CA, Gianni L (2011) Triple-negative breast cancer: an unmet medical need. Oncologist 16 Suppl 1: 1–11. 12. Oliveira AM, Ross JS, Fletcher JA (2005) Tumor suppressor genes in breast cancer: the gatekeepers and the caretakers. Am J Clin Pathol 124 Suppl: S16–28. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35566