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. 2016 Oct 25;7(43):69703-69717.
doi: 10.18632/oncotarget.11941.

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer

Sergey A Dyshlovoy  1   2   3 , Katharina Otte  1 , Winfried H Alsdorf  1 , Jessica Hauschild  1 , Tobias Lange  4 , Simone Venz  5 , Christiane K Bauer  6 , Robert Bähring  6 , Kerstin Amann  7 , Ramin Mandanchi  1 , Udo Schumacher  4 , Jennifer Schröder-Schwarz  4 , Tatyana N Makarieva  2 , Alla G Guzii  2 , Kseniya M Tabakmakher  2 , Sergey N Fedorov  2 , Larisa K Shubina  2 , Igor E Kasheverov  8 , Heimo Ehmke  6 , Thomas Steuber  9 , Valentin A Stonik  2 , Carsten Bokemeyer  1 , Friedemann Honecker  1   10 , Gunhild von Amsberg  1
Affiliations

Marine compound rhizochalinin shows high in vitro and in vivo efficacy in castration resistant prostate cancer

Sergey A Dyshlovoy et al. Oncotarget. .

Abstract

Development of drug resistance is an inevitable phenomenon in castration-resistant prostate cancer (CRPC) cells requiring novel therapeutic approaches. In this study, efficacy and toxicity of Rhizochalinin (Rhiz) - a novel sphingolipid-like marine compound - was evaluated in prostate cancer models, resistant to currently approved standard therapies. In vitro activity and mechanism of action of Rhiz were examined in the human prostate cancer cell lines PC-3, DU145, LNCaP, 22Rv1, and VCaP. Rhiz significantly reduced cell viability at low micromolar concentrations showing most pronounced effects in enzalutamide and abiraterone resistant AR-V7 positive cells. Caspase-dependent apoptosis, inhibition of pro-survival autophagy, downregulation of AR-V7, PSA and IGF-1 expression as well as inhibition of voltage-gated potassium channels were identified as mechanisms of action. Remarkably, Rhiz re-sensitized AR-V7 positive cells to enzalutamide and increased efficacy of taxanes.In vivo activity and toxicity were evaluated in PC-3 and 22Rv1 NOD SCID mouse xenograft models using i.p. administration. Rhiz significantly reduced growth of PC-3 and 22Rv1 tumor xenografts by 27.0% (p = 0.0156) and 46.8% (p = 0.047) compared with controls with an increased fraction of tumor cells showing apoptosis secondary to Rhiz exposure. In line with the in vitro data, Rhiz was most active in AR-V7 positive xenografts in vivo. In animals, no severe side effects were observed.In conclusion, Rhiz is a promising novel marine-derived compound characterized by a unique combination of anticancer properties. Its further clinical development is of high impact for patients suffering from drug resistant prostate cancer especially those harboring AR-V7 mediated resistance to enzalutamide and abiraterone.

Keywords: AR-V7; apoptosis; autophagy; castration resistant prostate cancer; rhizochalinin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1. Effect of Rhiz on viability and proliferation of prostate cancer cells
(A) The structure of Rhizochalinin (Rhiz, hydrochloride salt), derived from marine sponge Rhizochalina incrustata. (B) MTT assay: IC50 values (μM) of human prostate cancer cell lines treated with Rhiz for 48 h.
Figure 2
Figure 2. Effect of Rhiz on induction of apoptosis
Effect of Rhiz (0.5 and 1 μM) on induction of apoptosis (A) after 48 h of treatment in PC-3, DU145, 22Rv1, and VCaP cells, analyzed by FACS. Apoptotic cells were detected as sub-G1 population in the cell cycle distribution histogram; black columns indicate percentage of apoptotic cells at 1 μM of Rhiz (A). (B) Western blotting (cropped blots), showing dose-dependent cleavage of caspase-3 in prostate cancer cells at the indicated cell line dependent threshold concentrations of Rhiz to start caspase-3 cleavage after 48 h of treatment. (C) Western blotting (cropped blots) showing time-dependent cleavage of caspase-3 in prostate cancer cells treated with the indicated concentrations of Rhiz for 0.5, 1, 3, 24, and 48 h. Cells treated with 5 μM of anisomycin for 48 h (Aniso) were used as a positive control. (D), (E), Flow cytometry analyses of induction of apoptosis in PC-3 and DU145 cells treated with Rhiz using double staining annexin-V-FITC/PI. (D) PC-3 cells were treated with Rhiz at indicated concentrations for 6, 12, 24 or 48 h, and total apoptotic (annexin-V-FITC-positive) cells were quantified. (E) PC-3 and DU145 cells were pre-treated with 100 μM of pan-caspase inhibitor zVAD for 1 h following treatment with indicated concentrations of Rhiz for 48 h. Total apoptotic cells were quantified. (F) Western blotting analysis (cropped blots) of expression of pro- and anti-apoptotic protein expression in prostate cancer cells treated with Rhiz for 48 h.
Figure 3
Figure 3. Hallmarks of autophagy inhibition in PC-3 cells treated with Rhiz
(A) Effect of Rhiz on LC3B-I/II in PC-3 cells after 48 h of treatment. (B) Detection of autophagosome formation by electron microscopy. PC-3 cells were treated with Rhiz for 48 h. The number and size of autophagosomes (double membrane bound vesicles, indicated by yellow arrows) was significantly increased in treated cells. (C) Detection of autophagosomes by immunofluorescence microscopy. Cells were treated with 2 μM of Rhiz for 48 h, fixed, permeabilized, and treated with anti-LC3B-I/II antibody followed by treatment with Alexa Fluor 488-conjugated secondary antibody. The number of LC3B-I/II-positive organelles (autophagosomes, appeared as puncta and are indicated by arrows) is significantly increased in treated cells. (D), (E) Effect of Rhiz in combination with 3MA and BafA1 in PC-3 cells. Cells were co-treated with different concentrations of the single substances or their combination for 48 h. A constant molar ratio of the drugs ((D) ratio C(Rhiz):C(3MA) = 2:5000) or constant concentration of 10 nM BafA1 (E) was used. The combinational index (CI) values were calculated with CompuSyn Software. Cell viability was measured by MTT assay. (F), (G) Effect of Rhiz and other autophagy inhibitors and inducers on the LC3B-I/II level dynamics in PC-3 cells. Cells were exposed for 0.5 − 72 h to Rhiz, inhibitors of autophagy ((F) CQ, BafA1), or inducers of autophagy ((G) Rapa, starvation conditions – culture media was substituted with PBS). LC3B-I/II levels were detected by Western blotting (cropped blots).
Figure 4
Figure 4. Inhibitory effect of Rhiz on potassium channels
(A), (B) Effect of Rhiz in combination with potassium channel openers Minox (A) and Diaz (B) on viability of PC-3 and DU145 cells. Cells were co-treated with indicated concentrations of the drugs for 48 h. Cell viability was measured by MTT assay. (C–F) Effect of Rhiz on voltage-dependent potassium channels expressed in Xenopus oocytes. Currents mediated by heag1 (C) and Kv1.3 (D) and herg1 (F) are shown before and at the end of 10 μM Rhiz application. Dotted lines represent zero current. Pulse protocols are shown below traces. Time course and reversibility of current inhibition is shown for heag1 (C). (E), (F) Concentration-dependence of remaining current (% of control) in the presence of Rhiz for heag1 and Kv1.3 (E) and for herg1 ((F) outward and inward current) channels.
Figure 5
Figure 5. Effect of Rhiz on AR-V7, PSA, and IGF-1 protein and mRNA expression
(A) Effect of Rhiz on AR-V7 expression in 22Rv1 and VCaP cells. Protein expression was analyzed by Western blotting (cropped blots), mRNA levels were analyzed by qPCR. (B) Effect of Rhiz on PSA expression in 22Rv1 and LNCaP cells. The concentration of PSA in the culture supernatant was analyzed using ELISA and normalized to the number of viable cells, mRNA levels were analyzed by qPCR. (C) Effect of Rhiz on IGF-1 expression in 22Rv1, VCaP, and LNCaP cells analyzed by Western blotting (cropped blots), mRNA levels were analyzed by qPCR.
Figure 6
Figure 6. Effect of Rhiz on the cell viability in combination with other drugs
(A) Effect of Rhiz in combination with Enz in 22Rv1 and VCaP cells. Cell viability was measured by MTT assay. In all experiments cells were treated for 48 h. (B), (C) Effect of Rhiz in combination with docetaxel (B) or cabazitaxel (C) on PC-3, DU145 or 22Rv1 cells, examined by MTT assay. Cells were co-treated with different concentrations of the single substances or their combination for 48 h at a constant molar ratio. The combinational index (CI) values were calculated with CompuSyn software. The molar ratio of the substances used for the combination C(Rhiz):C(DOC) was 100:1 (for PC-3 and DU145 cells) or 25:1 (for 22Rv1 cells); for combination C(Rhiz):C(Caba) the molar ratio was 25:1 (for PC-3 cells) or 125:2 (for DU145 and 22Rv1 cells). The raw CI values from several independent experiments for each combination are represented on the correspondent graphs.
Figure 7
Figure 7. In vivo effect of Rhiz in a human prostate cancer xenograft model
PC-3 and 22Rv1 cells were injected subcutaneously into male NOD SCID mice. Once primary tumors reached 50–60 mm3, daily treatment (1.8 mg/kg/day) was started. Control group was treated with placebo (0.9% NaCl). Solutions were administrated i.p. (A) Effect of Rhiz on tumor growth. The lower bar graphs represent tumor masses measured at the day of termination of the experiments after the animals were sacrificed. (B) Detection of tumor cell death in vivo. Histological sections of representative tumors from control and treated animals were stained with H&E. Quantification of dead cells was performed using ImageJ Software. Tumor necrotic/apoptotic areas are indicated by arrows. (C) Induction of caspase-3 cleavage in PC-3 and 22Rv1 cell xenografts was analyzed by Western blotting using protein extracts of tumor samples. (D) Effect of Rhiz on body weight. (E) Effect of Rhiz treatment on white blood cell (WBC) count in PC-3 and 22Rv1 cell xenografts. (F) Effect of Rhiz treatment on spleen weight in PC-3 and 22Rv1 cell xenografts. (G), Model of the supposed mode of anticancer action of Rhiz: induction of caspase-dependent apoptosis possibly through the inhibition of potassium channels, inhibition of pro-survival autophagy and AR-signaling, and possible immuno-stimulatory effect.
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