薄荷精油抑制HRASQ61L突變角質細胞之活性及預防小鼠二階段皮膚致癌之功效

Abstract

流行病學統計顯示，大約20%到30%的黑色素瘤病人在給予BRAF抑制劑 (vemurafenib，PLX4032)的治療後會誘導繼發性皮膚鱗狀細胞癌或角化棘皮瘤的嚴重副作用。例如，在由化學藥劑7,12-dimethylbenz[a]anthracene (DMBA)以及12-O-tetradecanoylphorbol-13-acetate (TPA)誘導的二階段皮膚致癌模式中PLX4032會加速帶有HRAS突變基因的乳突狀瘤生成，而這個動物模式被認為用在模擬病人因PLX4032誘導產生的皮膚副作用是具有代表性的。本篇研究之目的為探討薄荷精油是否具有癌症化學預防的活性抑或預防PLX4032引起的皮膚副作用。研究中，確效了墾丁水薄荷精油及萊姆薄荷精油在測試的十種薄荷精油中具有最佳的抑制DMBA轉化的老鼠PDV角質細胞之細胞增生。同時也研究了萊姆薄荷精油中兩種最主要的化合物，檸檬烯(limonene)及香芹酮(carvone)合併使用的的生物活性。墾丁水薄荷精油、萊姆薄荷精油和檸檬烯+香芹酮抑制了受PLX4032刺激之PDV細胞的細胞群落生成能力，並誘導細胞週期停滯在G2/M期和引起細胞凋亡。PLX4032所促進PDV細胞的遷移及入侵能力，亦受到墾丁水薄荷精油、萊姆薄荷精油和檸檬烯+香芹酮之抑制，尤其萊姆薄荷精油具有最好的效果。而PLX4032促進PDV細胞中MAPK路徑的再活化也被抑制下來。我們進一步利用DMBA/TPA誘導的小鼠二階段皮膚致癌模式，FVB小鼠在有、無給予PLX4032下，探討墾丁水薄荷精油、萊姆薄荷精油和檸檬烯+香芹酮在生物體內的生物活性。在DMBA/TPA組，萊姆薄荷精油治療組在延緩乳突狀瘤的發生率及數量具有最好的效果。在另一實驗組中，我們觀察到PLX032會促進DMBA/TPA小鼠之乳突狀瘤的生成，而兩種薄荷精油和檸檬烯+香芹酮具延緩並抑制乳突狀瘤生成的效果。並且，受DMBA/TPA刺激之小鼠給予PLX，相較於兩種經由的治療，檸檬稀+香芹酮對於乳突狀瘤的生成具有較佳的抑制效果。在組織病理學檢驗之結果顯示，不論是否給予PLX4032，受DMBA/TPA刺激的皮膚皆會導致皮膚表皮層的增生，而墾丁水薄荷精油、萊姆薄荷精油和檸檬烯+香芹酮的治療均可改善這個現象，且乳突狀瘤組織中MAPK路徑的再活化現象被抑制且細胞凋亡現象亦被偵測出來。總結來說，此研究是第一次闡述墾丁水薄荷精油、萊姆薄荷精油和檸檬烯+香芹酮新穎的癌症化學預防作用，具有潛力預防BRAF抑制劑引起之皮膚副作用。

Epidemiological evidence shows that 20%-30% of cutaneous melanoma patients treated with the BRAF-inhibitor drug vemurafenib (PLX4032) develop secondary cutaneous squamous cell carcinomas or keratoacanthomas as a serious side effect. In a 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced two-stage skin carcinogenesis model, PLX4032 could accelerate the growth of skin papillomas harboring HRAS mutation. The model is considered to be a representative animal model that mimics the PLX4032-induced cutaneous side-effects in human patients. In this study, we aimed to investigate whether mint essential oils possess cancer chemoprevention activity and/or can prevent drug PLX4032-induced cutaneous side effects. We identified two essential oils from M. aquatica var. Kenting Water Mint (designated KWM-EO) and M. aquatica var. Citrata Lime Mint (designated LM-EO) among ten tested mint essential oils which exhibited significant anti-proliferation effect against PDV cells, a DMBA transformed mouse keratinocyte bearing HRASQ61L mutation cell line. The bioactivity of the combination of two major compounds present in LM-EO, i.e., limonene and carvone (designated L+C) was also evaluated. KWM-EO, LM-EO and L+C decreased colony formation, and induced G2/M cell-cycle arrest and cell apoptosis in the PLX4032-stimulated PDV cells. The migratory and invasive abilities of PDV cells promoted by PLX were also suppressed by KWM-EO, LM-EO and L+C treatments, among which LM-EO exhibited the highest activity. Paradoxical MAPK activation induced by PLX4032 in PDV cells was also inhibited by both EOs and L+C treatments. We further investigated the in vivo bioefficacy of both EOs and L+C combination treatments in the two-stage skin carcinogenesis animal model established using DMBA (initiator)/TPA (promoter) and FVB mice, with or without PLX4032 treatment. In the DMBA/TPA group, LM-EO treatment showed the best effect on reduction of papilloma incidence and total papilloma number in the DMBA/TPA-irritated mice. In a parallel study, the papilloma formation in the DMBA/TPA mice was found to be promoted by PLX4032 treatment and reversed by administration of LM-EO. KWM-EO and L+C treatments also attenuated the number of papillomas. Also, in the DMBA/TPA mice with PLX4032 treatment, L+C showed better inhibition of papilloma formation than the other two EOs. Histopathological examination showed that topical application of DMBA/TPA resulted in hyperplasia of the epidermis that was ameliorated by KWM-EO, LM-EO and L+C treatments. KWM-EO, LM-EO and L+C administration also diminished PLX-induced reactivation of MAPK signaling and promoted apoptosis in the DMBA/TPA stimulated papilloma tissues. Overall, this study is the first to demonstrate the novel cancer chemopreventive effect of essential oils from M. aquatica var. Kenting Water Mint and M. aquatica var. citrata Lime Mint, and the combination of limonene and carvone have great potential to prevent the cutaneous side effects induced by BRAF inhibitor drug PLX4032.