HYAL4-V1/Chondroitinase (Chase) Drives Gemcitabine Resistance and Predicts Chemotherapy Failure in Patients with Bladder Cancer.

PURPOSE: Gemcitabine-based chemotherapy regimens are first-line for several advanced cancers. Because of better tolerability, gemcitabine + cisplatin is a preferred neoadjuvant, adjuvant, and/or palliative chemotherapy regimen for advanced bladder cancer. Nevertheless, predicting treatment failure and overcoming resistance remain unmet clinical needs. We discovered that splice variant (V1) of HYAL-4 is a first-in-class eukaryotic chondroitinase (Chase), and CD44 is its major substrate. V1 is upregulated in bladder cancer and drives a malignant phenotype. In this study, we investigated whether V1 drives chemotherapy resistance. EXPERIMENTAL DESIGN: V1 expression was measured in muscle-invasive bladder cancer (MIBC) specimens by qRT-PCR and IHC. HYAL-4 wild-type (Wt) and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for chemoresistance and associated mechanism in preclinical models. RESULTS: V1 levels in MIBC specimens of patients who developed metastasis, predicted response to gemcitabine + cisplatin adjuvant/salvage treatment and disease-specific mortality. V1-expressing bladder cells were resistant to gemcitabine but not to cisplatin. V1 expression neither affected gemcitabine influx nor the drug-efflux transporters. Instead, V1 increased gemcitabine metabolism and subsequent efflux of difluorodeoxyuridine, by upregulating cytidine deaminase (CDA) expression through increased CD44-JAK2/STAT3 signaling. CDA inhibitor tetrahydrouridine resensitized V1-expressing cells to gemcitabine. While gemcitabine (25-50 mg/kg) inhibited bladder cancer xenograft growth, V1-expressing tumors were resistant. Low-dose combination of gemcitabine and tetrahydrouridine abrogated the growth of V1 tumors with minimal toxicity. CONCLUSIONS: V1/Chase drives gemcitabine resistance and potentially predicts gemcitabine + cisplatin failure. CDA inhibition resensitizes V1-expressing tumors to gemcitabine. Because several chemotherapy regimens include gemcitabine, our study could have broad significance.

Antitumor activity of sulfated hyaluronic acid fragments in pre-clinical models of bladder cancer.

Tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into angiogenic fragments (AGF: 10-12 disaccharides). AGF support tumor growth and progression. Urine and tissue HAase/HYAL-1 levels are sensitive markers for high-grade bladder cancer (BCa) and its metastasis. In preclinical models of BCa, we evaluated whether o-sulfated AGF (sHA-F) inhibits HAase activity and has antitumor activity. At IC50 for HAase activity inhibition (5-20 µg/ml [0.4-1.7 µM]), sHA-F significantly inhibited proliferation, motility and invasion of HYAL-1 expressing BCa cells (253J-Lung, HT1376, UMUC-3), P<0.001. sHA-F did not affect the growth of HYAL-1 non-expressing BCa (5637, RT4, T24, TCCSUP) and normal urothelial (Urotsa, SV-HUC1) cells. sHA-F treatment induced apoptosis by death receptor pathway. sHA-F downregulated transcript and/or protein levels of HA receptors (CD44, RHAMM), p-AKT, ß-catenin, pß-Catenin(S552), Snail and Twist but increased levels of pß-Catenin(T41/S45), pGSK-3α/ß(S21/S9) and E-cadherin. sHA-F also inhibited CD44/Phosphoinositide 3-kinase (PI-3K) complex formation and PI-3K activity. AGF addition or myristoylated-AKT overexpression attenuated sHA-F effects. Contrarily, HYAL-1 expression sensitized RT4 cells to sHA-F treatment. In the 253J-L and HT1376 xenograft models, sHA-F treatment significantly inhibited tumor growth (P<0.001), plausibly by inhibiting angiogenesis and HA receptor-PI-3K/AKT signaling. This study delineates that sHA-F targets tumor-associated HA-HAase system and could be potentially useful in BCa treatment.

The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer.

BIRM is an anticancer herbal formulation from Ecuador. Previous study established its antitumor and antimetastatic activity against prostate cancer models. The activity of BIRM against human prostate cancer (PCa) cells was investigated to uncover its mechanism of antitumor activity. In androgen receptor (AR)-expressing PCa cells BIRM was 2.5-fold (250%) more cytotoxic in presence of androgen (DHT) compared to cells grown in the absence of DHT. In AR-positive cells (LAPC-4 and LNCaP) BIRM caused a dose and time-dependent down-regulation of AR and increased apoptosis. Exposing cells to BIRM did not affect the synthesis of AR and AR promoter activity but increased degradation of AR via proteasome-pathway. BIRM caused destabilization of HSP90-AR association in LAPC-4 cells. It induced apoptosis in PCa cells by activation of caspase-8 via death receptor and FADD-mediated pathways. A synthetic inhibitor of Caspase-8 cleavage (IETD-CHO) aborted BIRM-induced apoptosis. The effect of BIRM on AKT-mediated survival pathway in both AR+ and AR- negative (PC-3 and DU145) showed decreased levels of p-AKTser 473 in all PCa cell lines. BIRM dosed by oral gavage in mice bearing PC-3ML tumors showed selective efficacy on tumor growth; before tumors are established but limited efficacy when treated on existing tumors. Moreover, BIRM inhibited the LNCaP tumor generated by orthotropic implantation into dorsal prostate of nude mice. Partial purification of BIRM by liquid-liquid extraction and further fractionation by HPLC showed 4-fold increased specific activity on PCa cells. These results demonstrate a mechanistic basis of anti-tumor activity of the herbal extract BIRM.

Dietary supplement 4-methylumbelliferone: an effective chemopreventive and therapeutic agent for prostate cancer.

BACKGROUND: Prevention and treatment of advanced prostate cancer (PCa) by a nontoxic agent can improve outcome, while maintaining quality of life. 4-methylumbelliferone (4-MU) is a dietary supplement that inhibits hyaluronic acid (HA) synthesis. We evaluated the chemopreventive and therapeutic efficacy and mechanism of action of 4-MU. METHODS: TRAMP mice (7-28 per group) were gavaged with 4-MU (450mg/kg/day) in a stage-specific treatment design (8-28, 12-28, 22-28 weeks). Efficacy of 4-MU (200-450mg/kg/day) was also evaluated in the PC3-ML/Luc(+) intracardiac injection and DU145 subcutaneous models. PCa cells and tissues were analyzed for HA and Phosphoinositide 3-kinase (PI-3K)/Akt signaling and apoptosis effectors. HA add-back and myristoylated Akt (mAkt) overexpression studies evaluated the mechanism of action of 4-MU. Data were analyzed with one-way analysis of variance and unpaired t test or Tukey's multiple comparison test. All statistical tests were two-sided. RESULTS: While vehicle-treated transgenic adenocarcinoma of the prostate (TRAMP) mice developed prostate tumors and metastases at 28 weeks, both were abrogated in treatment groups, without serum/organ toxicity or weight loss; no tumors developed at one year, even after stopping the treatment at 28 weeks. 4-MU did not alter the transgene or neuroendocrine marker expression but downregulated HA levels. However, 4-MU decreased microvessel density and proliferative index (P < .0001,). 4-MU completely prevented/inhibited skeletal metastasis in the PC3-ML/Luc(+) model and DU145-tumor growth (85-90% inhibition, P = .002). 4-MU also statistically significantly downregulated HA receptors, PI-3K/CD44 complex and activity, Akt signaling, and ß-catenin levels/activation, but upregulated GSK-3 function, E-cadherin, and apoptosis effectors (P < .001); HA addition or mAkt overexpression rescued these effects. CONCLUSION: 4-MU is an effective nontoxic, oral chemopreventive, and therapeutic agent that targets PCa development, growth, and metastasis by abrogating HA signaling.

Dietary supplement hymecromone and sorafenib: a novel combination for the control of renal cell carcinoma.

PURPOSE: Current treatments for metastatic renal cell carcinoma do not extend survival beyond a few months. Sorafenib is a targeted drug approved for metastatic renal cell carcinoma but it has modest efficacy. Hymecromone is a nontoxic dietary supplement with some antitumor activity at high doses of 450 to 3,000 mg per day. Hymecromone inhibits the synthesis of hyaluronic acid, which promotes tumor growth and metastasis. We recently noted that the hyaluronic acid receptors CD44 and RHAMM are potential predictors of metastatic renal cell carcinoma. In the current study we examined the antitumor properties of hymecromone, sorafenib and the combination in renal cell carcinoma models. MATERIALS AND METHODS: Using proliferation, clonogenic and apoptosis assays, we examined the effects of hymecromone (0 to 32 µg/ml), sorafenib (0 to 3.2 µg/ml) and hymecromone plus sorafenib in Caki-1, 786-O, ACHN and A498 renal cell carcinoma cells, and HMVEC-L and HUVEC endothelial cells. A Boyden chamber was used for motility and invasion assays. Apoptosis indicators, hyaluronic acid receptors, epidermal growth factor receptor and c-Met were evaluated by immunoblot. The efficacy of hymecromone, sorafenib and hymecromone plus sorafenib was assessed in the sorafenib resistant Caki-1 xenograft model. RESULTS: Hymecromone plus sorafenib synergistically inhibited proliferation (greater than 95%), motility/invasion (65%) and capillary formation (76%) in renal cell carcinoma and/or endothelial cells, and induced apoptosis eightfold (p <0.001). Hymecromone plus sorafenib inhibited hyaluronic acid synthesis and adding hyaluronic acid reversed the cytotoxicity of hymecromone plus sorafenib. Hymecromone plus sorafenib up-regulated pro-apoptotic indicators and down-regulated Mcl-1, CD44, RHAMM, phospho-epidermal growth factor receptor and phospho-cMet. In all assays hymecromone and sorafenib alone were ineffective. Oral administration of hymecromone (50 to 200 mg/kg) plus sorafenib (30 mg/kg) eradicated Caki-1 tumor growth without toxicity. Hymecromone and sorafenib alone were ineffective. CONCLUSIONS: To our knowledge this is the first study to show that the combination of sorafenib and the nontoxic dietary supplement hymecromone is highly effective for controlling renal cell carcinoma.

An orally active Amazonian plant extract (BIRM) inhibits prostate cancer growth and metastasis.

PURPOSE: Poor efficacy of conventional chemotherapeutic drugs against metastatic hormone-refractory prostate cancer (CaP) drives patients to try "alternative medicine". The antitumor activity of one such agent, "BIRM" (biological immune response modulator; "Simple Ecuadorian Oral Solution: an extract of an Amazonian plant"), was characterized in vitro and in vivo using established CaP cell lines and a tumor model. METHODS: The cytotoxicity of BIRM in four human and one rat CaP cell line was evaluated using cell proliferation-inhibition and clonogenic survival assays. BIRM-induced apoptosis, alterations in cell cycle phase progression and inhibition of the extracellular matrix-degrading enzyme hyaluronidase were also investigated in these cells. The in vivo efficacy of BIRM was evaluated in rats with subcutaneous tumor implants of Dunning EGFP-MAT LyLu cells. The active species in BIRM were characterized by gel filtration chromatography. RESULTS: BIRM inhibited cell proliferation and clonogenic growth of the CaP cells (IC(50) about 8.0 microl/ml). It increased cell accumulation in the G(0)/G(1) phase by 33.8% and decreased the proportion of cells in S phase by 54.6%. Apoptotic cell death in BIRM-treated cells was associated with activation of cell death-associated caspases. BIRM inhibited the activity of hyaluronidase, a hyaluronic acid-degrading enzyme, at 1 microl/ml. Treatment of MAT LyLu tumor-bearing rats with BIRM by oral gavage resulted in a significant decrease in tumor incidence (50%), tumor growth rate (18.6+/-1.3 days for 1 cc tumor growth in control rats and 25.7+/-2.6 days in BIRM-treated rats), and only one out of six BIRM-treated rats versus four out of six in the control group developed lung metastasis. Three active ingredients in BIRM with a relative molecular mass (Mr) of >or=3500 were identified by ultracentrifugation and gel filtration chromatography and were found to be resistant to proteinase and heat (100 degrees C). CONCLUSION: The plant extract BIRM contains antitumor compounds of Mr >or=3500 with potent antiproliferative activity in vitro and in vivo against prostate cancer cells.