Prostate Cancer Stem Cell-Targeted Efficacy of a New-Generation Taxoid, SBT-1214 and Novel Polyenolic Zinc-Binding Curcuminoid, CMC2.24

Background Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs. Principal Findings The CD133high/CD44high phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100nM-1µM; for 72 hr) induced about 60% cell death in CD133high/CD44+/high cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133high/CD44high cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days. Conclusions We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133high/CD44+/high tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 (“gene wake-up”), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs

The p53/p21 “gene-wake-up” induced by SBT-1214/CMC2.24 combination.

<p>(<b>A</b>) Previously absent pro-apoptotic/tumor suppressor proteins, p21 and p53 were induced by single treatment with SBT-1214/CMC2.24 combination for 24 hr in both CD133⁺ and bulk PPT2 cells. Such “gene wake-up” led to significant increase in the sensitivity of the CD133⁺ cells to this drug combination (B, C). Pre-treatment [SBT-1214 (1 µM) + CMC2.24 (10µM)]; second treatment [SBT-1214 (1 µM) + CMC2.24 variable]. Data were obtained with standard MMT assay.</p

The SBT-1214/CMC2.24 combination induced suppression of the stem cell-relevant transcription factors in CD133⁺ PPT2 cells.

<p>(<b>A</b>) Multiple stem cell-relevant transcription factors are up-regulated in the CD133+ cell population compared to their differentiated counterparts. (<b>B</b>) Down-regulation of the up-regulated transcription factors after treatment with 1 µM SBT-1214 and 10 µM CMC2.24 for 24 hr (PCR array assay; SABiosciences; PAHS 501). Western blot analysis confirmed significant down-regulation of key pluripotency transcription factors, c-Myc and Sox-2 in CD133⁺ cells (<b>C</b>). Histon H1 was used as a loading control.</p

Anti-tumor effects of SBT-1214 <i>in</i><i>vivo</i>.

<p>NOD/SCID mice were ectopically implanted with 3,000 of CD133⁺ PPT2 and PC3MM2 cells on the flanks. Three weeks after injection, mice were treated with weekly i.v. injections of SBT-1214 (x4: 40, 20, 20, 20 mg/kg). This treatment modality induced dramatic reduction in tumor size in the majority of the PPT2- and PC3MM2-induced tumors (<b>A</b>-<b>D</b>). Representative cases are shown on B and D. Values are the means ±SD; <i>p</i>≤0.0009 for PPT2-induced tumors (SBT-treated versus untreated controls; n=6), and <i>p</i>≤0.0018 for PC3MM2-induced tumors (n=6). Histopathological analysis of the residual tumors showed the presence of viable cells and accumulation of large multinucleated cells in 2 of 6 PPT2 tumors and 3 of 6 PC3MM2 tumors (representative H&E staining of untreated and SBT-1214-treated PPT2 tumor tissues is shown on <b>E, F</b>). <i>Ex </i><i>vivo</i> death of the drug-treated cells in culture (<b>G</b>). Control untreated tumor cells retained profound clonogenic and sphere-forming capacities during serial passaging (<b>H</b>).</p

Cytotoxic effects of the SBT-1214/CMC2.24 combination against prostate CD133⁺ cells.

<p>(<b>A</b>, <b>B</b>) The CMC2.24 as a single agent induced bi-phasic effects on prostate cancer cell proliferation: lower concentrations of it promoted proliferation, whereas higher ones were cytotoxic. (<b>C</b>) In contrast to SBT-1214, treatment with CMC2.24 did not induce an increase in the ratio of CD133⁺ cells (black dotted areas), but similarly to SBT-1214, increased expression of the differentiation marker pan-keratin (red dotted areas) and shifted the entire cell population toward differentiation (areas with asterisks). A combination of the two agents induced more significant cell death of the CD133⁺ PPT2 (<b>D</b>) and PC3MM2 (<b>E</b>) cells compared to each compound as a single agent. Data were obtained with standard MMT assay (<b>A</b>, <b>B</b>, <b>D</b>, <b>E</b>). Values are the means ±SD of the three independent experiments with 4 repeats for each drug concentration.</p

<i>In vitro</i> cytotoxicity of SBT-1214 and paclitaxel against prostate CSC-enriched cell populations.

<p>Representative FACS analysis shows a dose-dependent increase in the ratios of CD133^high PPT2 (<b>A</b>) and PC3MM2 (<b>B</b>) cells 24 hours after treatment with SBT-1214 and Ptx. Longer treatment (for 72 hours) with SBT-1214 (10 nM-10 µM) induced up to 65% cell death in PPT2 cells (<b>C</b>) and up to 60% in PC3MM2 cells (<b>D</b>; lower line). In contrast, treatment with the same doses of Ptx did not suppress proliferation of the tumorigenic prostate cancer cells (<b>C, D</b>; upper line). Cells were incubated with indicated drug concentrations, and data was obtained with standard MMT assay based on three independent experiments with four repeats in each treatment group. Values are the means±SD.</p

Molecular characterization of the primary prostate PPT2 cell line.

<p>(<b>A</b>) Representative FACS analyses of the different cell surface markers expression in unsorted PPT2 cells grown for 4 weeks on type I collagen in MSGB medium. Each dotted square represents the population of cells expressing moderate/high levels of a particular marker conjugated with different fluorescent tags. Note a long-term retention of the CD133-APC, standard (CD44-PE) and variant (CD44v6-FITC) CD44 and CD49f-APC. In contrast, only small fractions of the PPT2 cells expressed a marker of basal cells, p63, a marker of differentiated cells, pan-keratin, CK18, AR and CXCR4. (<b>B</b>) Immunohistochemical analysis shows that, in contrast to parental tumor tissue, purified CD133⁺ PPT2 cells do not express PSA <i>in </i><i>vitro</i>; however, PPT2-induced NOD/SCID tumor xenografts are weakly PSA-positive. (<b>C</b>) Immunocytochemical analysis shows uniform expression of vimentin and nestin, with especially high levels of these markers of neural stem cells in large MNCs. (<b>D</b>) Western blot analysis shows expression of the pluripotency markers in nuclear and cytoplasmic fractions of the CD133⁺ PPT2 cells. Both the nuclear and cytoplasmic fractions expressed c-Myc and were negative for p53 and p21; only the nuclear fraction expressed Oct-4 and Sox-2.</p