Rhodacyanine Derivative Selectively Targets Cancer Cells and Overcomes Tamoxifen Resistance

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family

Hsp70 Inhibition Reduces Akt and Selectively Kills Breast Cancer Cells

The family of 70-kDa proteins are molecular chaperones responsible for the noncovalent folding of many client proteins. including Akt,. Akt is a kinase client which is responsible for multiple anti-apoptotic and proliferation pathways. These oncogenic properties can be controlled by manipulating Hsp70 ATPase activity. By utilizing Hsp70 ATPase inhibitors we can reduce Akt levels in an Hsp70 dependent manner. These Akt reductions, coupled with the inhibition of the Hsp70 family of chaperones, yield cytotoxic events selective to cancer cells, specifically breast cancer. Here, we characterize two classes of Hsp70 family inhibitors, one a phenothiazine compound, methylene blue, as well as a novel rhodacyanine dye Ym1. Characterizations were conducted on the ER-positive breast cancer cell model MCF-7 cells, with functional readouts being LDH assays for toxicity, intracellular protein levels (Akt, ERa, etc.), as well as cellular localization. The goal of these experiments is to compare the effectiveness of these two Hsp70 inhibiting compounds. We aim to elucidate these two chemical backbones as potential targets for creating new compounds with both higher efficacy and specificity. The knowledge gained from understanding the biochemical properties of Hsp70 will demonstrate the viability of Hsp70 as a target in the treatment of cancer

Akt isoforms are concentration dependently reduced by YM-1, potential mechanism for resistance phenomena.

<p>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 ± SD (B).</p

Brief exposure to YM-1 restores tamoxifen effect in resistant cell model.

<p>TR-MCF7 cells were treated with 4-OHT for 48 hours, YM-1 (or vehicle) for 4 hours followed by 44 hours of either 4-OHT or 95% EtOH, or YM-1 (or vehicle) for 48 hours. At 48 hours from initial treatment, MTT viability assays were performed. Viability values of each treatment as a % of 48 hours of 4-OHT treatment ± SD (A). 2-Way ANOVA analysis comparing all YM-1 treatment groups (Gray squares- 4-hour YM-1then 4-OHT, open diamonds- 48 hour YM-1, black triangles- 4-hour YM-1 then 95% EtOH), revealed significance across concentrations (F(2,30) = 54.22, p<0.0001), and treatment strategy (F(4, 30) = 41.04, p<0.0001), but no significant interaction (F(8,30) = 1.83, p = 0.1107). * - indicates significant difference (p<0.05) of 4-hour YM-1 then 4-OHT from other two groups with exception of 10 µM treatments, significance as indicated (ns = p>0.05)(B). 1-way ANOVA of YM-1+4-OHT strategy revealing significance of 10 µM concentration (F(4,10) = 16.49, p = 0.0002)(C). Analysis of YM-1+95% EtOH, by 1-way ANOVA, revealed no significance across tested concentrations (F(4,10) = 3.435, p = 0.0516)(D). 48-hour YM-1 treatment showed significance differences between all concentrations and the 10 µM concentration, by 1-way ANOVA (F(4,10) = 12.32, p = 0.0007)(E). 1-way ANOVA analysis (F,(5,12) = 24.33, p<0.0001) comparing all 10 µM YM-1 treatments, 48-hour 4-OHT, and vehicle treatments revealed no significant difference between 48-hour 4-OHT and both 4-hour 10 µM YM-1+95% EtOH and 48-hour 10 µM YM-1 treatments; whereas the 48-hour 4-OHT and the 4-hour 10 µM YM-1+95% EtOH were significantly different from the 4-hour YM-1+4-OHT treatment (p<0.05)(F).</p

Phosphorylation but not localization of Estrogen Receptor α altered by YM-1.

<p>TR-MCF7 cells were treated with indicated conditions for 4 hours. Nuclear isolates and cytosolic fractions were compared by Western blot, representative blots shown (A & C). Densitometry analysis of pERα and ERα levels displayed as % of vehicle treatment ± SD (B & D).</p

TR-MCF7 cells and MCF7 cells susceptible to YM-1 toxicity at 48 hours but not at 4 hours; tamoxifen does not alter cytotoxicity.

<p>TR-MCF7 and parental MCF7 cells were treated for 48 hours in with 10 µM YM-1. After 48 hours, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment ± SD (A). MCF7 cells were treated for 4 hours with 10 µM YM-1. At 4 hours, medium was replaced with standard growth media for 44 hours. TR-MCF7 cells were treated with 10 µM YM-1 for 4 hours. At 4 hours, the media was removed and replaced with standard TR-MCF7 media containing 10 µM 4-OHT or 95% EtOH, the vehicle for 4-OHT, for 44 hours. After 48 hours from initial treatment, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment ± SD (B).</p

YM-1 toxicity specific to cancer cells; non-cancer cells unaffected.

<p>MCF7 cells (breast cancer) were treated for 24 hours with increasing concentrations of YM-1. After 24 hours, medium was collected and analyzed by LDH cytotoxicity assay. Values shown are a % of vehicle treatment ± SD (A). Hs578T and MDA-MB-231 (breast cancer), M17 (neuroblastoma), H4 (neuroglioma), and HeLa (cervical cancer) cell treated with increasing concentrations of YM-1 and the toxicity was compared to NIH-3T3 (mouse embryonic fibroblast) and HEK 293 (human embryonic kidney) cells for cancer specific toxicity. All cell lines were treated for 24 hours. After 24 hours, media were collected and analyzed by LDH assay. Values shown are a % of vehicle treatment ± SD (B).</p