Soesiawati R. Darjatmoko

Resveratrol Inhibits Tumor Growth of Human Neuroblastoma and Mediates Apoptosis by Directly Targeting Mitochondria

Purpose: Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Because resveratrol, a natural plant product, has been reported to have limited toxicity at chemotherapeutic levels, we investigated its efficacy in the treatment of neuroblastoma as well as its underlying mechanism of action. Experimental Design: Resveratrol was tested in mouse xenograft models of human neuroblastoma and in vitro using human cell lines. Results: Resveratrol inhibited the outgrowth of tumors by as much as 80%. The bioavailability of the drug in serum was in the low micromolar range (2-10 μmol/L) and no accumulation was observed in tumor tissue. When resveratrol levels were increased by peritumor injection, rapid tumor regression occurred. Resveratrol decreased tumor cell viability in vitro by 75% to 90%, resulting from an inhibition of cell proliferation and an induction of apoptosis. Loss of mitochondrial membrane potential was an early response to resveratrol. In addition, resveratrol treatment of isolated mitochondria also led to depolarization, suggesting that the drug may target mitochondria directly. Following depolarization, resveratrol caused the release of cytochrome c and Smac/Diablo from the mitochondria and subsequently the activation of caspase-9 (4- to 8-fold) and caspase-3 (4- to 6-fold). Conclusions: These studies indicate that, despite low bioavailability, resveratrol is effective at inhibiting tumor growth. Elevated levels of resveratrol enhance its antitumor potency leading to tumor regression, associated with widespread tumor cell death, the underlying mechanism of which involves the direct activation of the mitochondrial intrinsic apoptotic pathway.

Mitochondria, calcium, and calpain are key mediators of resveratrol-induced apoptosis in breast cancer.

Resveratrol (RES), a natural plant polyphenol, has gained interest as a nontoxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. However, the early molecular mechanisms of RES-induced apoptosis are not well defined. Using the human breast cancer cell lines MDA-MB-231 and MCF-7, we demonstrate that RES is antiproliferative and induces apoptosis in a concentration- and time-dependent manner. Preceding apoptosis, RES instigates a rapid dissipation of mitochondrial membrane potential by directly targeting mitochondria. This is followed by release of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) into the cytoplasm and substantial increase in the activities of caspases-9 and -3 in MDA-MB-231 cells. In addition, live cell microscopy demonstrates that RES causes an early biphasic increase in the concentration of free intracellular calcium ([Ca2+]i), probably resulting from depletion of the endoplasmic reticulum stores in breast cancer cells. In caspase-3–deficient MCF-7 cells, apoptosis is mediated by the Ca2+-activated protease, calpain, leading to the degradation of plasma membrane Ca2+-ATPase isoform 1 and fodrin; the degradation is attenuated by buffering [Ca2+]i and blocked by calpain inhibitors. Mitochondrial permeability transition pore antagonists also blocked calpain activation. In vivo mouse xenograft studies demonstrate that RES treatment inhibits breast cancer growth with no systemic toxicities. Together, these results suggest a critical role for mitochondria not only in the intrinsic apoptotic pathway but also in the Ca2+ and calpain-dependent cell death initiated by RES. Thus, RES may prove useful as a nontoxic alternative for breast cancer treatment.

Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB.

Resveratrol, a nontoxic natural product, exhibits multifaceted biological effects including antimutagenic and anticancer properties. We examined the effect of resveratrol on the expression and activation of Akt/protein kinase B and its impact on melanoma cell migration and invasiveness. We also explored the use of resveratrol as an antimalignant treatment option against skin melanoma in mouse models of the disease. Akt expression and activity were determined by a combination of real-time PCR and western blot analysis. Cell lines stably expressing Akt or a dominant negative variant were used to further establish the role of Akt during the response to resveratrol. Wound healing and transwell assays were used as in-vitro correlates of melanoma cell migration and invasiveness. The efficacy of resveratrol in the treatment of melanoma was assessed in two syngeneic mouse models. Resveratrol downregulated and inactivated Akt in B16F10 and B16BL6 melanoma cells. Resveratrol also inhibited the migratory and invasive properties of these highly malignant cells. The reduction of cell migration and invasion, however, was reversed in cell lines overexpressing Akt or after cotreatment with pharmacological inhibitors that blocked Akt degradation. Dominant-negative Akt cells were more sensitive to resveratrol and had diminished migratory properties. Oral treatment with resveratrol reduced primary tumor volume, Akt expression, and the propensity for metastasis in syngeneic mouse models of melanoma. These results suggest that resveratrol can reduce the malignant properties of highly invasive melanoma cells by inactivating Akt. The nontoxic targeting of Akt by resveratrol makes it an attractive treatment option for melanoma.

Resveratrol Metabolites Do Not Elicit Early Pro-apoptotic Mechanisms in Neuroblastoma Cells

Resveratrol, a nontoxic polyphenol, has been shown to inhibit tumor growth in a xenograft mouse model of neuroblasoma. However, resveratrol is rapidly metabolized, mainly to its glucuronidated and sulfated derivatives. This study demonstrates that resveratrol alone, and not the glucuronidated or sulfated metabolites, is taken up into tumor cells, induces a rise in [Ca(2+)](i), and ultimately leads to a decrease in tumor cell viability. A new water-soluble resveratrol formulation was delivered directly at the site of the tumor in a neuroblastoma mouse model. The amount of unmodified resveratrol associated with the tumor increased more than 1000-fold. The increase of unmodified resveratrol associated with the tumor resulted in tumor regression. The number of residual tumor cells that remained viable also decreased as the ratio of the metabolites relative to unmodified resveratrol declined.

Development of choroidal neovascularization in rats with advanced intense cyclic light-induced retinal degeneration.

OBJECTIVES To study the progressive changes of intense cyclic light-induced retinal degeneration and to determine whether it results in choroidal neovascularization (CNV). METHODS Albino rats were exposed to 12 hours of 3000-lux cyclic light for 1, 3, or 6 months. Fundus examination, fundus photography, fluorescein and indocyanine green angiography, and optical coherence tomography were performed prior to euthanization. Light-exposed animals were euthanized after 1, 3, or 6 months for histopathological evaluation. Retinas were examined for the presence of 4-hydroxy-2-nonenal- and nitrotyrosine-modified proteins by immunofluorescence staining. RESULTS Long-term intense cyclic light exposure resulted in retinal degeneration with loss of the outer segments of photoreceptors and approximately two-thirds of the outer nuclear layer as well as development of subretinal pigment epithelium neovascularization after 1 month. Almost the entire outer nuclear layer was absent with the presence of CNV, which penetrated the Bruch membrane and extended into the outer retina after 3 months. Absence of the outer nuclear layer, multiple foci of CNV, retinal pigment epithelial fibrous metaplasia, and connective tissue bands containing blood vessels extending into the retina were observed after 6 months. All intense light-exposed animals showed an increased presence of 4-hydroxy-2-nonenal and nitrotyrosine staining. Optical coherence tomographic and angiographic studies confirmed retinal thinning and leakiness of the newly formed blood vessels. CONCLUSIONS Our results suggest that albino rats develop progressive stages of retinal degeneration and CNV after long-term intense cyclic light exposure, allowing the detailed study of the pathogenesis and treatment of age-related macular degeneration. CLINICAL RELEVANCE The ability to study the progressive pathogenesis of age-related macular degeneration and CNV will provide detailed knowledge about the disease and aid in the development of target-specific therapy.

Ocular renin angiotensin: EM immunocytochemical localization of prorenin

Prorenin (PR) was localized by electron microscopic (EM) immunostaining of cryo-ultramicrotomy sections of human ciliary body and correlated with light microscopic immunostaining. Both layers of the ciliary epithelium contained the prohormone. However, density was much higher in the adjacent extracellular spaces, particularly in the vitreous cortex. This observation adds further evidence to a role of the ciliary epithelium in the transfer, storage or synthesis of components of a putative ocular renin angiotensin system.

Anti-tumor and immunomodulatory activity of resveratrol in vitro and its potential for combining with cancer immunotherapy

We evaluated the anti-tumor effect of Resveratrol (RV) on M21 and NXS2 tumor cell lines and its immunosuppressive activity on human and murine immune cells to determine the potential for combining RV and immunotherapy. In vitro, concentrations of RV≥25 mcM, inhibited cell proliferation, blocked DNA synthesis and induced G1 phase arrest in tumor and immune cells. RV at 12-50 mcM inhibited antibody dependent cell mediated cytotoxicity (ADCC) of tumor cells facilitated by the hu14.18-IL2 immunocytokine (IC). The in vivo anti-tumor and immunomodulating activity of RV given systemically were assessed in mice. Results showed that this RV regimen inhibited the growth of NXS2 tumors in vivo but did not appear to interfere with blood cell count, splenocyte or macrophage function. Thus, RV may be a candidate for combining with immunotherapy.

A Study of Histopathological Features of Latanoprost-Treated Irides With or Without Darkening Compared With Non–Latanoprost-Treated Irides

OBJECTIVES To study the histopathological features of latanoprost-treated irides with or without darkening, compared with non-latanoprost-treated irides. METHODS Iridectomy specimens and patient history forms were independently examined by 3 ophthalmic pathologists in a masked fashion. Specimens were evaluated for premalignant changes and for differences in level of pigmentation and degrees of cellularity, inflammation, and vascular abnormalities. RESULTS The specimens consisted of 22 latanoprost-treated darkened irides, 35 latanoprost-treated irides without darkening, and 35 non-latanoprost-treated irides. There was a statistically significant decrease in the number of nuclear invaginations and prominent nucleoli in latanoprost-treated darkened irides compared with the other 2 groups (P = .004 and P = .005, respectively). The average thickness and pigmentation of the anterior border layer was greater in the latanoprost-treated darkened irides than in the other 2 groups (P = .03 and P = .02, respectively). The latanoprost-treated darkened irides had increased pigmentation of the stroma (P < .001), stromal fibroblasts (P < .001), melanocytes (P = .005), vascular endothelium (P = .02), and adventitia (P < .001) relative to the other 2 groups. CONCLUSIONS There is no histopathological evidence of premalignant changes in latanoprost-treated darkened irides. The latanoprost-induced iris color changes are due to a thickening of the anterior border layer and an increased amount of melanin in the anterior border layer and within the stromal melanocytes.

The Sustained Delivery of Resveratrol or a Defined Grape Powder Inhibits New Blood Vessel Formation in a Mouse Model of Choroidal Neovascularization

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch’s membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

Use of Combination Therapy with Cisplatin and Calcitriol in the Treatment of Y-79 Human Retinoblastoma Xenograft Model

Background: Retinoblastoma is the most common primary malignant intraocular neoplasm of childhood. The poor outcomes of patients with metastatic retinoblastoma have encouraged the search for new therapies. In the current study, the efficacy of combination therapy with calcitriol and cisplatin in athymic mice with subcutaneous Y-79 human retinoblastoma tumours was assessed. Methods: 60 athymic mice were subcutaneously injected with human Y79 retinoblastoma cells. Animals were randomised into four groups: group 1, 50 μg of cisplatin; group 2, 0.05 μg of calcitriol; group 3, 0.05 μg of calcitriol and 50 μg of cisplatin; group 4, control. The cisplatin was administered once a week, and the calcitriol was given five times a week. Results: There was a significant inhibition of tumour growth in animals treated with the combination therapy of calcitriol and cisplatin as compared with controls and cisplatin alone (p = 0.0001 and p = 0.0041 respectively). In terms of toxicity, serum calcium levels were increased, but there was no mortality and minimal nephrotoxicity in any of the groups. Conclusion: The present study shows that cisplatin given in combination with calcitriol may be a viable multidrug therapy option in the treatment of high-risk retinoblastoma.