Topical 2′-Hydroxyflavanone for Cutaneous Melanoma

Abstract

2′-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towards multiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermal implants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growth of the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro. Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SK-MEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signaling proteins TNFα, and phospho-PDGFR-β were depleted in all three cell lines; MEKK-15 was depleted by 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-β inhibitor) and AZD 2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive, antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously been shown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evident from inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topically applied 2HF–Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumors implanted in mice and caused no overt toxicity despite significant systemic absorption. 2HF treatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas it increased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled by endocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF–PLO gel may be useful for topical therapy of cutaneous metastases of melanoma and could enhance the antineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF in melanoma overlaps with RLI76 inhibitors.

Rlip Depletion Suppresses Growth of Breast Cancer

“…Results demonstrated that the fluorescence was distributed throughout the cytoplasm of control antisense (CAS) or vehicle-treated cells, whereas in cells transfected with Rlip-LNA, the rhodamine signal remained localized near the plasma membrane ( Figure 5 B) as we showed previously [ 58 ]. The inhibitory effects of Rlip-LNA on endocytosis were also similar to those observed previously by depletion of Rlip by the R508 antisense and were consistent with previous studies showing that the endocytic process is interrupted soon after internalization, leaving vesicles stalled along the plasma membrane [ 38 , 58 ]. In conclusion, our results demonstrated that inhibition of EGF endocytosis and induction of apoptosis by Rlip depletion are possible mechanisms of anticancer effects in the human breast cancer cell lines MCF7 and MDA-MB-231.…”

“…We previously demonstrated that the rate of GS-E efflux by Rlip mutants correlates directly with its anti-apoptotic activity and with the rate of endocytosis of EGF and insulin [ 34 ]. We recently observed that depletion of Rlip protein also inhibits CDE in melanoma and lung cancer [ 58 , 59 ]. To determine whether LNA-mediated depletion of Rlip is also relevant in inhibition of CDE in breast cancer cells, we investigated the effect of Rlip depletion by antisense on the endocytosis of EGF-rhodamine.…”

“…These findings add breast cancer to the list of cancers susceptible to Rlip deficiency, joining melanoma, lung cancer, prostate cancer, colon cancer, renal cell carcinoma, pancreatic cancer, and neuroblastoma [ 27 , 34 , 37 , 38 , 39 , 40 , 41 , 58 , 60 ]. In addition to this remarkably wide spectrum of activity, an existential role of Rlip in cancer progression is supported by the failure of Rlip-knockout mice to develop cancer upon exposure to the powerful carcinogens benzo[a]pyrene and dimethylbenzanthracene, which cause cancer in 100% of wild-type mice [ 34 ].…”

“…For cell viability assays, cells were seeded at 1 × 10 5 cells/mL in DMEM and 100 µL/well in 96 well plates and allowed to recover for 16–18 h. The next day, one set of cells was transfected with 0.1 µg/well CAS, R508, or Rlip-LNA using Lipofectamine 3000, and another set of cells was treated with 20 µg/well control IgG, Rlip-polyclonal antibody, or Rlip-monoclonal antibody. After incubation at 37 °C for 48 h, cell proliferation was assayed as described previously using MTT [ 58 ]. For colony-forming assays, 1 × 10 5 cells / 500 µL were incubated in 10 µg/mL R508 or scrambled-antisense for 24 h, and then aliquots of 50 or 100 µL were added to 60 mm Petri dishes containing 4 mL culture medium.…”

Haploinsufficiency Interactions of RALBP1 and TP53 in Carcinogenesis

“…In stark contrast, mice lacking the RALBP1 ((RalA Binding Protein 1) gene, which encodes RLIP76 (also known as Rlip (RALBP1), used here for both gene and protein), a stress-responsive, anti-apoptotic protein, are highly resistant to carcinogenesis by even the most potent chemical carcinogens [ 10 ]. Targeted inhibition or depletion of RALBP1 causes regression of many types of cancer [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. In studies to examine the effect of combined RALBP1 and TP53 deficiency, we discovered that hemizygous RALBP1 deficiency was sufficient to exert a strong dominant negative effect on the spontaneous carcinogenesis phenotype of homozygous TP53-null mice [ 10 ].…”