Synthetic resveratrol analogue, 3,3',4,4',5,5'-hexahydroxy-trans-stilbene, accelerates senescence in peritoneal mesothelium and promotes senescence-dependent growth of gastrointestinal cancers

Abstract

3,3',4,4',5,5'-Hexahydroxy-trans-stilbene (M8) is a synthetic resveratrol derivative, advertised as a candidate drug highly effective against numerous malignancies. Because multiple tumors prone to M8 frequently metastasize into the peritoneal cavity, this study was aimed at establishing the effect of M8 on the growth and senescence of human peritoneal mesothelial cells (HPMCs), the largest cell population within the peritoneum, actively involved in the intraperitoneal spread of cancer. The study showed that M8, used at the highest non-toxic dose of 10 μM, impairs proliferation and accelerates senescence in cultured HPMCs via an oxidative stress-dependent mechanism. At the same time, soluble factors released to the environment by HPMCs that senesced prematurely in response to M8 promoted growth of colorectal and pancreatic carcinomas in vitro. These findings indicate that M8 may indirectly-through the modification of normal (mesothelial) cells phenotype-facilitate an expansion of cancer cells, which challenges the postulated value of this stilbene in chemotherapy.

Figure 1

The chemical structure of stilbene M8 (3,3′,4,4′,5,5′-hexahydroxy-trans-stilbene). Additional hydroxy (–OH) groups in the highly reactive position ortho are marked in the circles. The role of the ortho hydroxyl groups in the augmentation of some biological properties of resveratrol (RVT) derivatives has been explained in the discussion section.

Figure 2

Effect of M8 on viability and proliferation of early-passage human peritoneal mesothelial cells (HPMCs). Cell viability was examined using the MTT assay on the confluent cultures (A); Cell proliferative capacity was examined according to the measurements of cell distribution within the cell cycle, in particular in the S phase (darkened area) (B); and the percentage of cells positive for proliferating cell nuclear antigen, PCNA (C); The values shown in the panel B indicate the size of cell fraction in the S phase, in the control cells and the cultures exposed to 10 μM M8, respectively. Representative results of PCNA immunostaining in the control cells and the cultures exposed to 10 μM M8 (D). PCNA-positive cells exhibit brown nuclei. All experiments were performed on cell cultures derived from the first passage and exposed to M8 at 0.5 and 10 μM for 24 h. The asterisks indicate a significant difference compared to the control group. Experiments were performed in triplicates with HPMC (human peritoneal mesothelial cells) cultures derived from 8–12 different donors.

Figure 3

Effect of M8 on replicative senescence of HPMCs. Cells were forced to senescence by serial passaging at seven-day intervals, as described in the methods section, and then the cumulative number of population doublings (CPD) (A) and the activity of SA-β-Gal (B) were examined. A box in the panel (A) indicates the time-point at which M8-treated cells became senescent while the control cells still proliferated vigorously. The samples of conditioned medium were taken at this point and then used in the experiments shown in Figure 4. Representative results of early-passage HPMC staining for SA-β-Gal (positive cells have dark colour within the cytoplasm) (C). The asterisks indicate a significant difference compared to the control group. Experiments were performed in with HPMC cultures derived from 12 different donors.

Figure 4

The role of oxidative stress in M8-dependent premature senescence of HPMCs. The changes in 8-OHdG concentration in early-passage and senescent HPMCs subjected to M8 (A) as well as representative results of 8-OHdG immunostaining (positive cells have dark nuclei) in early-passage cultures (B); The results of time-course experiments for reactive oxygen species (ROS) production (C) and superoxide dismutase (SOD) activity (D) in early-passage cells exposed to M8; The effect of ROS scavenger, N-tert-butyl-α-phenylnitrone (PBN, at 800 μM), on the activity of SA-β-Gal in cells treated with 10 μM M8 (E). The cells were plated at a low density and then subjected to the stilbene for up to 72 h. The asterisks indicate a significant difference compared to the control group while the symbol # indicates a significant difference compared with M8-treated cells. Experiments were performed in duplicates with HPMCs derived from 8–10 different donors.

Figure 5

Effect of M8 on HPMC-dependent proliferation of ovarian (A2780), colorectal (SW480), and pancreatic (PSN-1) cancer cells. Cell proliferation was assessed on low-density cultures exposed for 24 h to the conditioned medium harvested from HPMCs that senesced prematurely in response to 10 μM M8 and to the medium from the control cells (from the same passage), using [³H]-thymidine incorporation assay (A); The measurements of cancer cell distribution within the cell cycle, in particular in the S phase (darkened area) (B); The values shown in the panel (B) indicate the size of cell fraction in the S phase, in the cultures exposed to CM (conditioned medium) from control HPMCs and to CM from HPMCs prematurely senesced in response to 10 μM M8, respectively. The representative results of western-blot analysis of cell cycle regulators: cyclins D and E. Bands corresponding to target molecules were compared to those for GAPDH (C). The asterisks indicate a significant difference compared to the control cells treated as 100%. The experiments were performed in quadruplicates with CM from HPMC cultures obtained from 5 different donors.