Bioactivity of betulinic acid nanoemulsions on skin carcinogenesis in transgenic mice K14E6

1. INTRODUCTION

Cancer is one of the main causes of death worldwide. It is characterized by a deregulation of cell growth and proliferation (Diaz-Cano, 2015Diaz-Cano SJ. 2015. Pathological bases for a robust application of cancer molecular classification. Int. J. Mol. Sci. 16, 8655-8675. https://doi.org/10.3390/ijms16048655 ). It is estimated that by 2025 the incidence per year will reach 19.3 million (Bishayee and Block, 2015Bishayee A, Block K. 2015. A broad-spectrum integrative design for cancer prevention and therapy: The challenge ahead. Semin. Cancer Biol. 35, S1-S4. https://doi.org/10.1016/j.semcancer.2015.08.002 ) and it has been documented that melanoma-type skin cancer is associated with higher morbidity in Latino populations (Rodríguez et al., 2018Rodríguez VM, Shuk E, Arniella G, González CJ, Gany F, Hamilton JG, Gold GS, Hay JL. 2018. A qualitative exploration of Latinos’ perceptions about skin cancer : The role of gender and linguistic acculturation. J. Cancer Educ. 32, 438-446. https://doi.org/10.1007/s13187-015-0963-4.A ). In addition, the increase in the incidence of skin cancer is mainly attributable to prolonged periods of sun exposure. Firsthand treatments continue to be mainly radiotherapy, chemotherapy, surgery, hormone therapy and targeted therapies. However, these strategies have proven to show adverse secondary side-effects such as damage to adjacent tissues. Because of its incidence, finding alternative therapies to treat it is of utmost importance (Pejin et al., 2014Pejin B, Kojic V, Bogdanovic G. 2014. An insight into the cytotoxic activity of phytol at in vitro conditions. Nat. Prod. Res. 28 (22), 2053-2056. https://doi.org/10.1080/14786419.2014.921686 ). The use of natural compounds as a complementary therapy has attracted great interest in the prevention and treatment of cancer and since several biochemical and genetic mechanisms are involved in the development of cancer, it is necessary to investigate the different pathways in order to propose novel treatments (Palumbo et al., 2013Palumbo MO, Kavan P, Miller WH, Panasci L, Assouline S, Johnson N, Cohen V, Patenaude F, Pollak M, Jagoe RT, Batist G. 2013. Systemic cancer therapy: Achievements and challenges that lie ahead. Front. Pharmacol. 4 MAY, 1-9. https://doi.org/10.3389/fphar.2013.00057 ; Pejin et al., 2013Pejin B, Jovanovic K, Mojovic M, Savic A. 2013. New and Highly Potent Antitumor Natural Products from Marine-Derived Fungi: Covering the Period from 2003 to 2012. Curr. Top. Med. Chem. 13 (21), 2745-2766. https://doi.org/10.2174/15680266113136660197 ). Betulinic acid (3β, LUP-hydroxy-20 (29) -en-28-oic) is a triterpene isolated mainly from the bark of the white birch tree (Betula alba), to which several therapeutic properties have been attributed, with the most promising being its antitumoral effect. It has been reported to induce apoptosis in melanoma cells since 1995 (Fulda and Kroemer, 2009Fulda S, Kroemer G. 2009. Targeting mitochondrial apoptosis by betulinic acid in human cancers. Drug Discov. Today 14, 885-890. https://doi.org/10.1016/j.drudis.2009.05.015 ) by the loss of mitochondrial membrane potential through the release of cytochrome C, smac/DIABLO, the activation of caspases 3 and 9 by APAF1 binding among other proteins and, by causing DNA damage (Ali-Seyed et al., 2016Ali-Seyed M, Jantan I, Vijayaraghavan K, Bukhari SNA. 2016. Betulinic Acid: Recent Advances in Chemical Modifications, Effective Delivery, and Molecular Mechanisms of a Promising Anticancer Therapy. Chem. Biol. Drug Des. 87, 517-536. https://doi.org/10.1111/cbdd.12682 ). However, there are insufficient studies to explain the interaction between BA and cell cycle progression (Chintharlapalli et al., 2011Chintharlapalli S, Papineni S, Lei P, Pathi S, Safe S. 2011. Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors. BMC Cancer 11, 371. https://doi.org/10.1186/1471-2407-11-371 ). It has also been reported that BA induces cell cycle arrest in the G2/M phase by regulation of the Hiwi protein and Cyclin B (Yang et al., 2010Yang LJ, Chen Y, Ma Q, Fang J, He J, Cheng YQ, Wu QK. 2010. Effect of betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric adenocarcinoma AGS cells. Acta Pharmacol. Sin. 31, 66-72. https://doi.org/10.1038/aps.2009.177 ). BA has very little pharmacokinetic value due to its low aqueous solubility (~21 µg/mL) (Saneja et al., 2018Saneja A, Arora D, Kumar R, Dubey RD, Panda AK, Gupta PN. 2018. Therapeutic applications of betulinic acid nanoformulations. Ann. N.Y. Acad. Sci. 1421, 1-14. https://doi.org/10.1111/nyas.13570 ). Nanotechnology-based drug delivery systems in cancer therapy have been demonstrated to be suitable for the administration of lipophilic compounds by improving distribution capacity (Jabir et al., 2012Jabir NR, Tabrez S, Ashraf GM, Shakil S, Damanhouri GA, Kamal MA. 2012. Nanotechnology-based approaches in anticancer research. Int. J. Nanomedicine 7, 4391-4408. https://doi.org/10.2147/IJN.S33838 ). Nanoemulsions are characterized by their droplet size ranging between 20-200 nm (Kabri et al., 2011Kabri TH, Arab-Tehrany E, Belhaj N, Linder M. 2011. Physico-chemical characterization of nano-emulsions in cosmetic matrix enriched on omega-3. J. Nanobiotechnology 9, 41. https://doi.org/10.1186/1477-3155-9-41 ). The main focus of this research is based on assessing the effects of free and nanoencapsulated BA on a skin cancer mouse model.

2. MATERIALS AND METHODS

Phospholipase PLA₁ Lecitase^® Ultra was provided by NOVO (Salem, VA); Duolite A568 support was a gift from Rohm & Haas (Barcelona, Spain). Phosphatidylcholine (PC) (95% PC soy) was purchased from Shenyang Tianfeng Bioengineering Technology Co. (Shenyang, Liaoning, China). Free medium-chain fatty acids were obtained from the saponification of commercial Original Thin Oil^® (Dover, Idaho). Betulinic acid was purchased from Indofine Chemicals (Hillsborough, NJ). Fifteen K14E6 transgenic mice, descendent from the original mice provided by Prof. Patricio Gariglio, and five control FVB mice were provided by the animal facility of the Universidad Cristóbal Colón in Veracruz. The iTaqTM Universal SYBR Green One-Step (BIO RAD, Hercules, CA) Kit was employed for the RT-PCR assay.

3. RESULTS

3.1. Incorporation of free medium-chain fatty acids (MCFAs) to PC

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Free fatty acids were obtained as a product of the saponified medium-chain oil with a composition of 68.93% caprylic acid (C8:0) and 30.77% capric acid (C10:0). Adsorption of the enzyme onto the support was quantified by Bradford obtaining an immobilization of 64%, with 46 mg of protein adsorbed/g of support from an initial suspension of 75 mg of protein/g. Figure 1 shows the kinetics of the incorporation of MCFAs to PC (59%), the yield of PC (47%) and modified PC (86%) during 12 h of reaction.

Figure 1.  Incorporation of MCFAs, yield of PC and modified PC by acidolysis with PLA₁; Molar ratio of substrates (PC/MCFAs) of 1:15 and 12% enzyme concentration at 50 °C. PC= phosphatidylcholine, MCFAs= medium-chain fatty acids. Tukey’s test was used to compare averages in the incorporation of MCFAs, PC Yield and modified PC Yield. Each data point represents the mean of three replicates and their SD.

3.4. Effect of BANE on skin carcinogenesis

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In the sick control group, the number of tumors were 7.33 ± 5.8. In the group with the carcinogens and free BA, the mean number of tumors was 2.25 ± 1.25, which represents a 70% reduction, as shown in Figure 2. Finally, in the group treated with the carcinogens and BANE, no skin tumorigenesis was observed. In Table 3, the average tumor surface areas by groups are depicted compared with the healthy control group (FVB).

Figure 2.  Effect of free BA and BANE on skin tumorigenesis of K14E6 mice with DMBA (25 nmol/0.2 mL acetone) and TPA (6.8 nmol/0.2 mL acetone) for eight weeks. One-way ANOVA, P=0.007. Data points are mean values from five replicates; errors bars represent the SD.

Table 3.  Effect of BA on tumor surface area of all groups (mm²).

Healthy control (FVB)	Sick Control N/T	Free BA group	BANE group
0.00	15.62 ± 0.78A	14.92±0.97A	0.00±0.00B

Data represent the mean ± SD on each treated group (n= 5), One-way ANOVA/Tuckey P=0.002. Free BA-Free betulinic acid, BANE-betulinic acid nanoemulsión.

3.5. Histological evaluation

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Figure 3 illustrates the histological results of all 4 treatment groups: (A) healthy control group (FVB), a normal cell architecture is observed in the epidermis, dermis, subcutaneous tissue and appendages. The tissue of the sick control group (only treated with DMBA/TPA) (D), had moderately differentiated squamous keratinizing carcinoma development, composed entirely of atypical squamous epithelial cells with intense keratin production, neoplastic cells with irregular nuclei and abundant mitosis. In the group treated with free BA (G), the mice developed a keratinizing epidermoid carcinoma. The group treated with BANE (J), showed normal skin architecture without tumor lesions of any type and regular morphology was observed. The epidermal structure of the BANE-treated group was similar to the healthy control group.

Figure 3.  (a) Macroscopic appearance of healthy control; (b) histological dissection of skin without histological alterations (H&E, 2x); (c) histological dissection without alterations in hair follicles and sebaceous glands (H&E, 5x); (d) sick control macroscopic appearance of mouse with multiple epidermoid carcinomas; (e) infiltrating epidermoid carcinoma with areas of surface keratinization (H&E, 5x); (f) neoplastic cells with moderate cytoplasm, pleomorphic nuclei of granular chromatin and abundant mitoses (H&E, 40x); (g) macroscopic aspect of mice treated with free BA, shows two neoplastic lesions in the dorsal region, the largest of which is ulcerated; (h) neoplastic lesion corresponds to infiltrating epidermoid carcinoma with differentiation regions (H&E, 2x); (i) morphological appearance of neoplastic cells with broad keratinizing cytoplasm, large nuclei with dispersed granular chromatin, note also mitosis; (j) macroscopic aspect of mice treated with BANE, does not show neoplasia; (k) histological cut of skin without histological alterations (H&E, 2x); (l) histological dissection with slight chronic inflammatory infiltrate in the dermis (H&E, 10x).

3.6. Gene expresión

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The gene expression analysis (Figure 4) showed similar statistical RQ values for each treatment group: Cdk4 and Casp8. In the free BA and BANE groups, differences were evident when compared to the healthy and sick control groups; however, there was no difference between their signaling pathway level. RQ values were 0.03 and 0.0002 for Cdk4 and Casp8, respectively; the values for free BA and BANE were 0.04 and 0.0005, respectively. Cdk4 and Casp8 expression levels were reduced when free BA and BANE were supplied, suggesting that the mechanism of action of this compound may influence the cyclin-dependent pathway and repressed Rb1.

Figure 4.  Relative Quantitation values for Cdk4 and Casp8 gene expression. Student t-test was employed to calculate the levels of significance, and P < 0.05 was considered statistically significant. The data points represent the mean of five replicates and the error bar is the SD.

4. DISCUSSION

This investigation describes the bioactivity of free BA compared to its nanoemulsified form as well as the advantage of the enzymatic modification that was carried out through the use of phospholipase PLA₁ (Ochoa-Flores et al., 2017Ochoa-Flores AA, Hernandez-Becerra JA, Cavazos-Garduño A, Soto-Rodriguez I, Sanchez-Otero MG, Vernon-Carter EJ, Garcia HS. 2017. Enhanced Bioavailability of Curcumin Nanoemulsions Stabilized with Phosphatidylcholine Modified with Medium Chain Fatty Acids. Curr. Drug Deliv. 14, 377-385. https://doi.org/10.2174/15672018136661609191428 ). Our results showed an increase in the modification of PC with MCFAs under the optimal conditions mentioned in the results section. Recent research showed that the enzymatic modification of phospholipids by PLA₁ reached a significant level after 24 h of reaction (Li et al., 2014Li X, Chen JF, Yang B, Li DM, Wang YH, Wang WF. 2014. Production of structured phosphatidylcholine with high content of DHA/EPA by immobilized phospholipase A1-catalyzed transesterification. Int. J. Mol. Sci. 15, 15244-15258. https://doi.org/10.3390/ijms150915244 ). The choice of oil is an important variable, since it directly affects the NE droplet size, along with its chemical and optical properties (Cavazos-Garduño et al., 2015Cavazos-Garduño A, Ochoa Flores AA, Serrano-Niño JC, Martínez-Sanchez CE, Beristain CI, García HS. 2015. Preparation of betulinic acid nanoemulsions stabilized by ω-3 enriched phosphatidylcholine. Ultrason. Sonochem. 24, 204-213. https://doi.org/10.1016/j.ultsonch.2014.12.007 ). Stability increases with small sizes by avoiding gravitational segregation, flocculation by Brown’s movement and greater system transparency; their bio-accessibility has been increased in in vitro systems when using O/W NE (Salvia-Trujillo et al., 2013Salvia-Trujillo L, Qian C, Martín-Belloso O, McClements DJ. 2013. Influence of particle size on lipid digestion and b-carotene bioaccessibility in emulsions and nanoemulsions. Food Chem. 141, 1475-1480. https://doi.org/10.1016/j.foodchem.2013.03.050 ). It has been reported that using polylactide-co-glycolide-monomethoxy polyethylene glycol nanoparticles containing BA, where 147 nm globule sizes were obtained, increased its antitumor activity by up to 7 times in an Erlich carcinoma model (Saneja et al., 2017Saneja A, Kumar R, Singh A, Dhar Dubey R, Mintoo MJ, Singh G, Mondhe DM, Panda AK, Gupta PN. 2017. Development and evaluation of long-circulating nanoparticles loaded with betulinic acid for improved anti-tumor efficacy. Int. J. Pharm. 531, 153-166. https://doi.org/10.1016/j.ijpharm.2017.08.076 ). The hepatoprotective activity of BA in nanoemulsions with sizes of 150.3 nm, a PDI of 0.192, a Ȥ-potential of -10.2 mV has been reported, together with an increase in its solubility when dispersed in olive oil in Wistar rats with CCl₄ as a toxic agent (Harwansh et al., 2017Harwansh RK, Mukherjee PK, Biswas S. 2017. Nanoemulsion as a novel carrier system for improvement of betulinic acid oral bioavailability and hepatoprotective activity. J. Mol. Liq. 237, 361-371. https://doi.org/10.1016/j.molliq.2017.04.051 ). In the same way, using chicken embryos, the anti-angiogenic effect of nanoemulsions with a globule size of 145 nm and a Ȥ-potential of -39.1 mV has been documented (Dehelean et al., 2011Dehelean CA, Feflea S, Ganta S, Amiji M. 2011. Anti-angiogenic effects of betulinic acid administered in nanoemulsion formulation using chorioallantoic membrane assay. J. Biomed. Nanotechnol. 7, 317-24. https://doi.org/10.1166/jbn.2011.1297 ). In this study, particle sizes of 40 nm, a PDI of 0.14, and a Ȥ-potential of -15 mV were attained. The reduction in particle size increased the superficial area, enhancing the absorption capacity of the bioactive by biological membranes such as the intestinal lining and/or skin; i.e., it improved the transport, exposure, and activity of the encapsulated compound, evidencing that a minimum amount can be employed to exert its therapeutic activity. The use of carrier systems has opened a field to investigate the different routes of the carcinogenesis process in murine models. This has been envisioned for the development of drugs that use natural compounds with low bioavailability, such as in the case of BA. In a research regarding skin damage monitoring, the anti-angiogenic process and the damage caused to blood vessels by TPA and DMBA exposure to BALB/c mice were described; betulin was used for 12 weeks and produced a reduction in blood vessels; and no tumor development was observed (Dehelean et al., 2011Dehelean CA, Feflea S, Ganta S, Amiji M. 2011. Anti-angiogenic effects of betulinic acid administered in nanoemulsion formulation using chorioallantoic membrane assay. J. Biomed. Nanotechnol. 7, 317-24. https://doi.org/10.1166/jbn.2011.1297 ). When evaluating BA (both topic and oral NE) in C57BL/6J mice, with a liver implanted with B6 melanoma cells, a decrease in metastasis was found to be significant compared to the non-treated group (Ciurlea et al., 2010Ciurlea SA, Dehelean CA, Ionescu D, Berko S, Csanyi E, Hadaruga DI, Ganta S, Amiji MM. 2010. A comparative study regarding melanoma activity of Betulinic acid on topical ointment vs. systemic nanoemulsion delivery systems. J. Agroaliment. Processes Technol. 16, 420-426.). When a complex of cyclodextrin with BA (100 mg/kg) was intraperitoneally administered in an in vivo melanoma model (C57BL/6J) for 21 days, the tumor volume decreased from 300 to 150 mm³. However, the administration method, even though it was effective, caused significant pain and distress to the animal. Because of the BA structure, it can be metabolized and degraded, thereby releasing the compound before the target cells could be reached (Soica et al., 2014Soica C, Danciu C, Savoiu-Balint G, Borcan F, Ambrus R, Zupko I, Bojin F, Coricovac D, Ciurlea S, Avram S, Dehelean CA, Olariu T, Matusz P. 2014. Betulinic acid in complex with a gamma-cyclodextrin derivative decreases proliferation and in vivo tumor development of non-metastatic and metastatic B164A5 cells. Int. J. Mol. Sci. 15, 8235-8255. https://doi.org/10.3390/ijms15058235 ). In this research, we prepared a nanoemulsion system to be administered orally, without causing harm to the animal. Moreover, it was formulated with nutraceutical materials that provide additional health benefits, such as MCFAs and lecithin. These are important components in the architecture of cell membranes which increase the bioavailability of the bioactive compound and enhance its intestinal absorption by enterocytes, or it is absorbed directly through the lymphatic system. One of the advantages of our nanoemulsified system is the globule size of < 200 nm, allowing better absorption of the compound through cellular membranes. Also, an additional benefit of using MCFAs is the increased bio-accessibility of lipophilic compounds; unlike long-chain fatty acids, MCFAs are more easily dispersed in aqueous phases and confer better protection to the nanoemulsion against lipolytic enzymes; they can also form micelles in the intestine for the solubilization of hydrophobic bioactive compounds. Furthermore, it is hypothesized that the chemical structure of BA resembles cholesterol in terms of its insertion into the plasmatic membrane. It was estimated that BA changed cell membrane fluency, and thereby modulated the signaling pathway associated with membrane microdomains, translocated certain receptors, and inhibited cell proliferation and migration (Chen et al., 2016Chen CL, Chen CY, Chen YP, Huang YB, Lin MW, Wu DC, Huang HT, Liu MY, Chang HW, Kao YC, Yang PH. 2016. Betulinic acid enhances TGF-ß signaling by altering TGF-ß receptors partitioning between lipid-raft/caveolae and non-caveolae membrane microdomains in mink lung epithelial cells. J. Biomed. Sci. 23, 1-15. https://doi.org/10.1186/s12929-016-0229-4 ). It has also been proposed that its antitumor effect is caused by the inhibition of tumor angiogenesis, promoting a decrease in the supply of nutrients and oxygen to potential tumor formations; therefore, a decrease in growth rate was probably caused by low rates of cell necrosis (Aisha et al., 2012Aisha AFA, Abu-Salah KM, Ismail Z, Malik A, Abdul S. 2012. α-Mangostin Enhances Betulinic Acid Cytotoxicity and Inhibits Cisplatin Cytotoxicity on HCT 116 Colorectal Carcinoma Cells. Molecules 17, 2939-2954. https://doi.org/10.3390/molecules17032939 ), but also increased cellular apoptosis, loss of membrane potential and cell cycle arrest (Saneja et al., 2017Saneja A, Kumar R, Singh A, Dhar Dubey R, Mintoo MJ, Singh G, Mondhe DM, Panda AK, Gupta PN. 2017. Development and evaluation of long-circulating nanoparticles loaded with betulinic acid for improved anti-tumor efficacy. Int. J. Pharm. 531, 153-166. https://doi.org/10.1016/j.ijpharm.2017.08.076 ). The specific cytotoxicity of BA has been previously found in neuroblastoma and glioblastoma cell lines (Thurnher et al., 2003Thurnher D, Turhani D, Pelzmann M, Wannemacher B, Knerer B, Formanek M, Wacheck V, Selzer E. 2003. Betulinic acid: A new cytotoxic compound against malignant head and neck cancer cells. Head Neck. 25, 732-740. https://doi.org/10.1002/hed.10231 ); however, the mechanism is not yet fully understood. Implicated genes in skin and cervical cancer have been outlined to clarify these conditions and determine whether BA can be effective to counter it. In that study, Ccne2 and Cdk4 were upregulated in skin cancer and showed no changes in cervical cancer. This suggested that the above genes were involved in these particular cancer types (Mendoza-Villanueva et al., 2008Mendoza-Villanueva D, Diaz-Chavez J, Uribe-Figueroa L, Rangel-Escareão C, Hidalgo-Miranda A, March-Mifsut S, Jimenez-Sanchez G, Lambert P, Garglio P. 2008. Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice. BMC Cancer 8, 347. https://doi.org/10.1186/1471-2407-8-347 ). It is important to note that the transgenic mouse model used in our project, K14E6, was designed to express E6 and E7 genes from Human Papilloma Virus (HPV) in skin, promoting the development of skin cancer; these genes were implanted in the embryo’s ectoderm for their future expression. It has been reported that E6 inhibits p53, which is a tumor suppressor gene that regulates cell cycle by mediating p21. Additionally, E7 inhibits the expression of the retinoblastoma (RB) gene, another tumor suppressor gene that also regulates cell cycle. Under normal circumstances both p53 and RB cease cell division at the G₁ phase by compromising CDK binding to Cyclins or by restricting DNA replication, respectively. This prevents the progression to the S phase when there is a defect in cell division; conversely, in this case both genes were under-expressed, producing defective and uncontrolled proliferation of skin cancer cells. Casp8 expression levels were also found to be down-regulated in skin cancer, producing a favorable environment for the progression of this type of cancer. Attenuation of apoptosis attributed to Caspase inhibition has been suggested as one of the mechanisms for tumorigenesis onset in several types of cancer. In this manner, alteration in the Caspase expression might be a diagnostic biomarker for skin carcinoma. In our research, subjects treated with BA showed a significant decrease in Cdk4 when compared to our control untreated mice. This biomarker has been attributed as a target for the BA potential therapeutic effect by arresting the cell cycle at the G₁ phase as expected, as well as inducing apoptosis in melanoma cell lines. In this type of cancer Cdk4 is upregulated (Rieber and Rieber, 2006Rieber M, Rieber MS. 2006. Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 status. Int. J. Cancer 118, 1135-1143. https://doi.org/10.1002/ijc.21478 ). It has been proposed that BA selectively activates the intrinsic apoptotic pathway in cancer cells by releasing cytochrome C, activating caspase 3 but not caspase 8; BA also regulates members of the Bcl-2 family and induces apoptosis in HeLa cells directly through this intrinsic pathway (Fulda, 2009Fulda S. 2009. Betulinic acid: A natural product with anticancer activity. Mol. Nutr. Food Res. 53, 140-146. https://doi.org/10.1002/mnfr.200700491 ; Gali-Muhtasib et al., 2015Gali-Muhtasib H, Hmadi R, Kareh M, Tohme R, Darwiche N. 2015. Cell death mechanisms of plant-derived anticancer drugs: Beyond apoptosis. Apoptosis. 20, 1531-1562. http://doi.org/10.1007/s10495-015-1169-2 ). BA bearing dephosphorylates the 3-kinase (PI3K)/Akt phosphatidylinositol pathway by promoting cell viability in cancer cells, thereby repressing Cdk inhibitors p27 and the p21 cell cycle progression modulator. BA has proven to cease cell cycle progression in cell lines such as HeLa, at the G0/G1 phase through the inhibition of the PI3K/Akt pathway by incubating 30 μmol/L BA after 12 hours; in addition, it was found to contribute to mitochondrial apoptosis by membrane depolarization and increased Caspase activity (Xu et al., 2017Xu T, Pang Q, Wang Y, Yan X. 2017. Betulinic acid induces apoptosis by regulating PI3K/Akt signaling and mitochondrial pathways in human cervical cancer cells. Int. J. Mol. Med. 40, 1669-1678. http://doi.org/10.3892/ijmm.2017.3163 ).

The results described in this research suggest the enhanced beneficial effect of BA in its nanoemulsified form as a protector against skin carcinogenesis development and progression, as it can mediate multiple pathways as well as intervene in cell cycle regulation. It is still necessary to better clarify the interaction of BA in both its free and nanoencapsulated forms directly on the cell cycle, in order to explain the favorable response that was observed in this work, and thus be able to propose its use as a complementary therapy.

5. CONCLUSIONS

A complete inhibition of tumor development was observed in mice treated with BANE, confirming its antitumor activity in vivo, which is consistent with previous studies performed by several authors. Our data suggest a clear advantage of nanoencapsulating BA over its application in its free form. However, further studies remain to be carried out in order to precisely elucidate the molecular mechanisms, gene regulation, and cell cycle interference achieved by the BANE.