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

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

<p>Abstract</p> <p>Background</p> <p>Growing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133^high/CD44^highfraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity <it>in vivo </it>and <it>in vitro</it>, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133^high/CD44^highcolon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214.</p> <p>Results</p> <p>Selected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (<it>SA</it>Biosciences) revealed that colonospheres induced by purified CD133^high/CD44^highexpressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 μM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities.</p> <p>Conclusions</p> <p>We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133^high/CD44^highcells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors <it>in vivo </it>may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.</p

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

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