Alternative Options for Skin Cancer Therapy via Regulation of AKT and Related Signaling Pathways

Global environmental pollution has led to human exposure to ultraviolet (UV) radiation due to the damaged ozone layer, thereby increasing the incidence and death rate of skin cancer including both melanoma and non-melanoma. Overexpression and activation of V-akt murine thymoma viral oncogene homolog (AKT, also known as protein kinase B) and related signaling pathways are major factors contributing to many cancers including lung cancer, esophageal squamous cell carcinoma and skin cancer. Although BRAF inhibitors are used to treat melanoma, further options are needed due to treatment resistance and poor efficacy. Depletion of AKT expression and activation, and related signaling cascades by its inhibitors, decreases the growth of skin cancer and metastasis. Here we have focused the effects of AKT and related signaling (PI3K/AKT/mTOR) pathways by regulators derived from plants and suggest the need for efficient treatment in skin cancer therapy.


Introduction
Skin cancer is one of the most frequent cancers worldwide [1,2], with increasing annual costs and morbidity [3,4]. Based on their cellular origin, skin cancers are mainly divided into melanoma (derived from melanocytes) and non-melanoma skin cancer (NMSC, from epithelial cells). NMSC includes basal cell carcinoma (~80%) and squamous cell carcinoma (~16%) and melanoma accounts for~4% of all skin cancer [5]. Even though it accounts for a small portion of skin cancer, melanoma is the most malignant cause of invasive metastasis with a five year survival rate of~20% [6]. An estimated 7,700,000 new cases of NMSC, including 5,900,000 basal cell carcinomas and 1,800,000 squamous cell carcinomas, have been reported globally including 195 countries accounting for 65,000 cancer deaths [2,7]. An estimated 100,350 new cases of melanoma and 6850 related deaths have been reported in the USA during 2020 [1]. The major environmental risk factors for skin cancer (melanoma and NMSC) include UVA, UVB and UVC [6,8]. UVC has the shortest wavelengths spectrum (100-280 nm) and after absorption by the ozone layer and atmosphere, it barely reaches the earth. Generally, the human skin is affected   (GS)/cyclin D1 for protein synthesis, cell survival, cell cycle, metabolism, migration and invasion ( Figure 1). The other hands, PTEN/PI3K/AKT-mediated pathways promote cancer cell migration and invasion by the upregulation of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF) [24,25]. Therefore, it is desirable to regulate the AKT kinase expression and activation and related signaling pathways for skin cancer therapy.

AKT and Related Signaling Pathway Inhibitors for Skin Cancer Regulation
Numerous phytomedicines derived from natural plant or fruit extracts exhibit anticancer activities against cell proliferation, survival, migration, metastasis and angiogenesis in vitro and in vivo. It has been previously reported that acacetin, isoangustone A, sulforaphane and tryptanthrin inhibited melanoma cell proliferation and tumor growth, and induced cell cycle arrest and apoptosis by directly or indirectly targeting PI3K/AKT/mTOR signaling pathways [26][27][28][29]. Here we describe the mechanisms of the latest natural inhibitors targeting AKT kinase and related signaling pathways in skin cancer preclinical studies in vitro and in vivo (Tables 1 and 2, Figure 3).

AKT and Related Signaling Pathway Inhibitors for Skin Cancer Regulation
Numerous phytomedicines derived from natural plant or fruit extracts exhibit anticancer activities against cell proliferation, survival, migration, metastasis and angiogenesis in vitro and in vivo. It has been previously reported that acacetin, isoangustone A, sulforaphane and tryptanthrin inhibited melanoma cell proliferation and tumor growth, and induced cell cycle arrest and apoptosis by directly or indirectly targeting PI3K/AKT/mTOR signaling pathways [26][27][28][29]. Here we describe the mechanisms of the latest natural inhibitors targeting AKT kinase and related signaling pathways in skin cancer preclinical studies in vitro and in vivo (Tables 1 and 2, Figure 3).

Polyphyllin I
Diosgenyl alpha-L-rhamnopyranosyl-(1-2)-(beta-L-ara-binofuranosyl-(1-4)-beta-D-glucopyranoside), is a major component of Paris polyphylla and inhibits the growth of gastric and ovarian cancers, and osteosarcoma [34,35]. Treatment with polyphyllin I at 1.5, 3.0 and 6.0 mg/L concentrations suppressed A375 melanoma cell progression by reducing cell proliferation, migration and invasion, and the induction of G1 cell cycle accumulation, apoptosis and autophagy at 48 h [36]. Polyphyllin I-induced apoptosis and autophagy was mediated via the downregulation of pPI3K, pAKT and pmTOR expression. It was validated by A375 cell xenograft mouse experiments demonstrating that polyphyllin I (5 mg/kg, i.p.) inhibited tumor growth in both volume and weight. The levels of proliferation marker, Ki-67 and apoptosis feature, TUNEL-positive cells, were inhibited and increased in polyphyllin I-treated tissues compared with vehicle-treated controls, respectively.

Sinomenine
The alkaloid component 7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinane-6-one is derived from Sinomenium acutum which is used for rheumatoid arthritis [43]. The anticancer activities of sinomenine have been reported in ovarian, gastric and lung cancers [31,44]. Sinomenine significantly inhibited caspase-3 activity and the viability of B16-F10 mouse melanoma cells and induced apoptosis and related proteins expression (Bax/Bcl-2 ratio) at 25, 50 and 100 µM in a CCK-8 assay and Annexin V stained cell counting as well as in Western blot analysis [45]. In detail, sinomenine increased autophagy by the induction of beclin1 expression and LC3II/LC3I level ratio and reduction in p-p62/SQSTM1 expression by the inhibition of pAKT and pmTOR expression, and thereby suppressed the cell growth and increased apoptosis/autophagy. The results were confirmed by treatment of cells with chloroquine, an autophagy inhibitor and a LY294002, PI3K/AKT inhibitor. Moreover, sinomenine (100 mg/kg, s.c.) retarded the melanoma tumor growth in vivo.

Ginkgo Biloba Exocarp Extract
Derived from Ginkgo biloba L. (Ginkgo nuts) via the water solution-alcohol precipitation method, suppressed Lewis lung cancer cell growth or angiogenesis by regulation of mitogen-activated protein kinase (MAPK) signaling pathways and Wnt/β-catenin-vascular endothelial growth factor (VEGF) signaling pathways [49,50]. Ginkgo biloba exocarp extract inhibited B16-F10 cell proliferation, migration and adhesion to HUVEC cells in vitro at 10, 20 and 40 µg/mL by decreasing the expression of pPI3K and pAKT, and reduced B16F10 cells grafted tumor growth and lung metastasis in C57BL/6J mice, in vivo at 50, 100 and 200 mg/kg [51].

Other Bioactive Components
The viability of human uveal melanoma UM-1 cells was inhibited by treatment with pristimerin, a quinine methide triterpenoid compound derived from Celastraceae and Hippocrateaceae and the apoptosis induction was mediated by disrupting the mitochondrial membrane potential and increasing the ROS production. Furthermore, pristimerin reduced migration and invasion by the regulation of pAKT and pFoxO3a expression with confirming the knock-down of AKT in UM-1 cells [61]. In human A375 and mouse B16F10 melanoma cells, bioactive compounds such as gambogic acid [62], melittin [63], kaempferol [64], euplotin C [65], lycorine [66], oxyfadichalcone C [67], isoliquiritigenin [68], muniziqi granule/harmine [69], apigenin [70] and casticin [71] inhibited cell proliferation, colony formation, migration and invasion by downregulating the expression of pPI3K, pAKT, pmTOR and pGSK3β together with the expression of related biomarkers including p27, cyclin D1, LC3, 4EBP1, Bax, Bcl-2 and MMPs. A375.S2 cells, which are investigated in studies involving metastasis, chrysin [72] and berberine [73], significantly reduced cell mobility, migration and invasion by decreasing the level of MMPs, N-cadherin and uPA expression through the inhibition of PKC and pAKT. In A431 non-melanoma skin cancer cells, treatment with caffeic acid n-butyl ester induced G2/M phase of cell cycle arrest and apoptosis and inhibited migration by decreasing the expression of pPI3K, pAKT and pmTOR [74].

Perspectives
Multisteps of skin carcinogenesis are processed by initiation, promotion and progression [75]. UV is itself both initiator and promoter, and chemicals such as DMBA and TPA are initiators or initiator/promoters. Initiators trigger the DNA damage or ROS production. It can be removed by repair system in healthy cells, however, when the cells fail to recover from DNA damage or oxidative stress then the cells are transformed into neoplastic cells. This initiation and promotion steps indicated by inflammation markers including COX-2, NF-kB and AP-1, and PI3K/AKT/mTOR signaling pathways. Transformed cells continuously progress to cancer by proliferation and spread to other organs by migration and invasion. In these stages, PI3K/AKT/mTOR signaling pathways mediate to induce the survival and migration/invasion biomarkers including cyclins, Bcl-2 family and MMPs. Phytochemicals derived from plants can control each step of carcinogenesis, cancer proliferation and metastasis. In Tables 1 and 2, most of natural compounds showed antiproliferation, antisurvival, antimigration and anti-invasion of skin cancers by the regulation of AKT-mediated signaling while syringic acid, herbacetin and α-mangostin inhibited DMBA/TPA or UV-induced skin carcinogenesis. Herbacetin directly targeted the ATP-binding pocket of the AKT catalytic domain and others are indirectly affected by AKT up-or downstream signaling pathways. Most effective phytochemicals contain the structure of flavonoid and polyphenols; however, it is difficult to dissolve in water and therefore limited to effect on target organs in vivo. Therefore, the formulation of natural compounds for improving the permeability could be modified such as ethosome. Binary ethosome of evodiamine and fisetin derived from Evodia rutaecarpa and onion enhanced the inhibitory activities of B16 melanoma cell proliferation and UVB-induced inflammation in mice [76,77]. Additionally, the combination therapy of clinical agents and natural compounds is expected to yield positive results. Application of natural compounds in skin cancer therapy requires the standardization of the plant-derived components and elucidation of their action mechanisms. On the other hand, we should consider other options with natural compounds mediated not only via canonical AKT-mediated signaling pathways but also new AKT-mediated signaling mechanisms such as miR-152-3p/c-MET/AKT and AKT/PFKFB4 pathways [31,33].