Neomenthol prevents the proliferation of skin cancer cells by restraining tubulin polymerization and hyaluronidase activity

Abstract

Introduction: Neomenthol, a cyclic monoterpenoid, is a stereoisomer of menthol present in the essential oil of Mentha spp. It is used in food as a flavoring agent, in cosmetics and medicines because of its cooling effects. However, neomenthol has not been much explored for its anticancer potential. Additionally, targeting hyaluronidase, Cathepsin-D, and ODC by phytochemicals is amongst the efficient approach for cancer prevention and/or treatment.

Objectives: To investigate the molecular and cell target-based antiproliferative potential of neomenthol on human cancer (A431, PC-3, K562, A549, FaDu, MDA-MB-231, COLO-205, MCF-7, and WRL-68) and normal (HEK-293) cell lines.

Methods: The potency of neomenthol was evaluated on human cancer and normal cell line using SRB, NRU and MTT assays. The molecular target based study of neomenthol was carried out in cell-free and cell-based test systems. Further, the potency of neomenthol was confirmed by quantitative real-time PCR analysis and molecular docking studies. The in vivo anticancer potential of neomenthol was performed on mice EAC model and the toxicity examination was accomplished through in silico, ex vivo and in vivo approaches.

Results: Neomenthol exhibits a promising activity (IC₅₀ 17.3 ± 6.49 μM) against human epidermoid carcinoma (A431) cells by arresting the G2/M phase and increasing the number of sub-diploid cells. It significantly inhibits hyaluronidase activity (IC₅₀ 12.81 ± 0.01 μM) and affects the tubulin polymerization. The expression analysis and molecular docking studies support the in vitro molecular and cell target based results. Neomenthol prevents EAC tumor formation by 58.84% and inhibits hyaluronidase activity up to 10% at 75 mg/kg bw, i.p. dose. The oral dose of 1000 mg/kg bw was found safe in acute oral toxicity studies.

Conclusion: Neomenthol delayed the growth of skin carcinoma cells by inhibiting the tubulin polymerization and hyaluronidase activity, which are responsible for tumor growth, metastasis, and angiogenesis.

Keywords: AA, Arachidonic acid; AKLP, Alkaline phosphatase; Ab/Am, Antibiotic/antimycotic; BE, Binding energy; BIL, Bilirubin total & direct; BSA, Bovine serum albumin; BUN, Blood urea nitrogen; CATD, Cathepsin D; CHOL, Cholesterol; CM-H2DCFDA, Chloromethyl derivative of dichloro fluorescin diacetate; COX-2, Cyclooxygenase 2; CRTN, Creatinine; Cancer biomarker; DCFDA, 2′,7′ dichloro fluorescin diacetate; DFMO, α-difluoro methyl ornithine; DHFR, Dihydrofolatereductase; DMEM, Dulbecco’s minimal essential media; DMSO, Dimethyl sulfoxide; DNA, Deoxyribonucleic acid; DOXO, Doxorubicin; EAC, Ehlrich Ascites Carcinoma; EC50, Half maximal effective concentration; EDTA, Ethylene diamine tetra acetic acid; ELISA, enzyme-linked immunosorbent assay; Ehrlich Ascites Carcinoma; FACS, Fluorescence-Activated Cell Sorting; FBS, Fetal bovine serum; FDA, Food and Drug Administration; FOX, Ferrous oxidation-xylenol orange; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase, HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; HA, Hyaluronic acid; HDAC, Histone deacetylase; HDL, High density lipoprotein; HYAL, Hyaluronidase; Human epidermoid carcinoma; Hyaluronidase; IC50, Half maximal inhibitory concentration; IDT, Integrated DNA Technologies; Ki, Inhibitory constant; LDH, Lactate dehydrogenase; LOX-5, Lipoxygenase-5; MEF, Mean erythrocyte fragility; MMP, Mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTX, Methotrexate; NAC, N-acetyl cysteine; NADPH, Nicotinamide adenine dinucleotide phosphate hydrogen; NRU, Neutral red uptake; NaOH, Sodium hydroxide; Neomenthol; ODC, Ornithine decarboxylase; OECD, Organization for Economic Co-operation and Development; OF, Osmotic fragility; PBS, Phosphate buffer saline; PCR, Polymerase chain reaction; PDB, Protein Data Bank; PDT, Podophyllotoxin; PEP A, pepstatin A; PI, Propidium iodide; PI3K, Phosphotidyl inositol-3 kinase; PKB/Akt, Protein kinase B; RBC, Red blood cell; RIPA, Radio immune precipitation assay buffer; RNA, Ribonucleic acid; RNase A, Ribonuclease A; ROS, Reactive oxygen species; RPMI, Roswell park memorial institute; Rh123, Rhodamine 123; SGOT, Aspartate aminotransferase; SGPT, Alanine aminotransferase; SRB, Sulphorhodamine B; TCA, Tricarboxylic acid; TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine; TNBS, Trinitrobenzenesulphonic acid; TPA, 12-O-Tetradecanoylphorbol-13-acetate; TPR, Total protein; TRIG, Triglyceraldehyde; TRPM8, Transient receptor potential member 8; Tubulin; URIC, Uric acid; WBC, White blood cell; mTOR, Mammalian target of rapamycin.

Graphical abstract

Fig. 1

Cell cycle analysis results of neomenthol in different cancer cell lines by using flow cytometry. PC-3, A431, FaDu, K562, MDA-MB-231, and A549 were treated with indicated concentrations of neomenthol for 24 h and stained with PI to determine DNA fluorescence and cell cycle distribution as described in material and methods section. Data were analyzed by FACS Diva software for the proportions of different cell cycle phases. The fraction of cells from apoptosis, G1, S and G2 phases analyzed from PE-A vs cell counts are shown in (%). Data expressed as mean ± SD. The arrest and apoptosis of each cell line was also represented in column. Cell specific standards were used i.e. doxorubicin (DOXO) was used for MDA-MB-231, PC-3, A431, taxol was used for A549 and podophyllotoxin (PDT) was used for FaDu and K562.

Fig. 2

Neomenthol inhibits tubulin polymerization. Neomenthol was incubated with general tubulin buffer (GTB) at indicated concentration and the kinetic study was performed by using UV–Vis spectrophotometer. Paclitaxel (PAC) was used as tubulin stabilizer and podophyllotoxin (PDT) was used as tubulin destablizer.

Fig. 3

Neomenthol increases ROS level in A431 cell line. (A) The DCFH-DA staining was used to detect ROS production in A431 cell line at indicated concentrations and analyzed by flow cytometry (B) flow cytometry results (C) A431 cells were treated with neomenthol at indicated concentration in 96-well plate for 24 h then incubated with 10 µM DCF-DA, analyzed by spectrofluorometer. Doxorubicin was used as the positive control. The data are presented as mean ± SD. Neomenthol was compared with control using one way ANOVA via Dunnett test through Graphpad Instat Software. (Non-significant changes were observed).

Fig. 4

Neomenthol decreases the mitochondrial membrane potential of A431 cell line. (A) Neomenthol pre-treated A431 cells were incubated with 10 µM Rh123, within 1 h analyzed by flow cytometry (B) flow cytometry results (C) A431 cells were treated with neomenthol at indicated concentration in 96-well plate for 24 h then incubated with 10 µM Rh123, analyzed by spectrofluorometer. Doxorubicin was used as the positive control. The data presented are mean ± SD. Neomenthol was compared with control using one way ANOVA via Dunnett test through Graphpad Instat Software (Non-significant changes were observed).

Fig. 5

Neomenthol modulates cell proliferation pathways in A431 cell line. A431 cells were incubated with the indicated dose of neomenthol for 24 h then crude protein from these cells was collected using lysis buffer. Estimation was done as per the instructions given in ELISA kit manual (PI3K, AKT, mTOR, HDAC-6, X axis). Doxorubicin (DOXO) was used as the positive control. The data was estimated as ng/mg of protein (Y Axis). All values are in mean ± SD. Treated samples were compared with control using one way ANOVA via Dunnett test through Graphpad Instat Software. (Non-significant changes were observed).

Fig. 6

EAC tumor reduction by neo-menthol in Swiss albino mice. One day after tumor induction in mice (i.p.), neomenthol was administrated intra-peritoneal to the animals for 9 days and tumor was evaluated on day 13. NaCl (0.9%) was given as vehicle to the control group and 5FU (5 fluorouracil) was used as the positive control. (A) Tumor volume, weight and inhibition (B) Tumor cells (C) Percent survival (D) Body weight and (E) Microscopic images of EAC cells. Data are expressed in mean ± SE (n = 5) and comparison was made between control groups and treated groups using one way ANOVA with Student t-test (^**p < 0.01 and ^***p < 0.001).

Fig. 7

Summary of neomenthol in modulating the expression of initiation (PI3K, AKT, mTOR, Tubulin), promotion (HDAC-6, COX-2), and progression (hyaluronidase) phase biomarkers in A431 cells. In cancer cells, upside (↑) arrow showed higher expression of biomarkers and downside arrow (↓) depicts the decrease of biomarker expression by neomenthol.