In vitro anti-melanoma activity and in vivo hair growth stimulation by Lophocereus marginatus methanol extracts

1. Introduction

Hair loss is one of the common effects of cancer treatment, due to the disruption of the hair growth cycle in the anagen or catagen phase by chemotherapy, which leads to excessive hair loss (Natarelli et al., 2023). Hair loss may be related to different causes, including chemotherapy, genetic predisposition, nutritional deficiency (vitamins, minerals, and poor diet), skin problems, drugs, poorly prepared aesthetic procedures, hormonal problems, stress, anxiety, and inflammatory processes (Gokce et al., 2022). Cancer is a pro-inflammatory multifactorial disease, particularly relevant in cutaneous melanoma. Due to melanoma folliculotropism, it may occur on the head, directly affecting hair growth, which is the reason for developing alopecia, either due to the different treatments or due to its pathophysiological process (Bedogni and Paus, 2020). Currently, the search for new treatments for hair growth has focused on stimulating its growth or preventing it (Mai et al., 2023). However, the side effects of treatments have been marginally investigated due to their functionality, as is the case with 6-(1-piperidinyl)-2,4-pyrimidinediamine 3-oxide sulfate (minoxidil) and the natural product bergamot oil from the plant Citrus bergamia. The use of natural products has been effective in promoting hair growth or improving the absorption of other products such as minoxidil (Wall et al., 2022). The Mexican cactus Lophocereus marginatus has been studied for the treatment of several types of cancer (Gomez-Flores et al., 2019). In addition, other reports have shown the presence of β-sitosterol and palmitic acid, which may promote hair growth and prevent its loss, according to in silico and in vivo studies (Zamani et al., 2022). The present study aimed to evaluate the in vitro antitumor effect of L. marginatus MeOH extracts against B16F10 murine melanoma cells and their in vivo potential to stimulate hair growth in the murine Balb/c model.

2. Materials and Methods

3. Results

3.1 In vitro effect of L. marginatus MeOH extract, minoxidil, and bergamot oil on tumor and normal cell growth

We determined the in vitro antitumor activity against the melanoma cell line B10F16 and the normal VERO cell line, and the in vivo hair-growth promoting effect of L. marginatus MeOH extract, minoxidil, and bergamot oil. As shown in Fig. 1, the L. marginatus MeOH extract and bergamot oil caused significant (p < 0.05) tumor cell growth inhibition, as compared with that of normal cells (bergamot oil was non-toxic to VERO cells at any concentrations evaluated). Minoxidil was highly toxic (> 85% growth inhibition) against B16F10 and VERO cells (Fig. 1). Furthermore, the SI of the L. marginatus extract was calculated for the B10F16 line, obtaining an SI of 3.7 and 3.56, as compared with NIH/3T3 and VERO cells, respectively (Table 2).

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Fig. 1.

Cell growth inhibition by (a) crude L. marginatus (Lm) methanol extract, (b) minoxidil, and (c) bergamot oil against B16F10 and VERO cell lines. Data represents the means ± SD of the percentage of growth inhibition at different treatment concentrations.

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Table 2 IC50 of L. marginatus MeOH extract against tumor and normal cell lines.

Treatment	IC50 (μg/mL)		SI
	B16F10	VERO
Lm extract	62.63 ± 3.3a	222.7 ± 6.4	3.7/3.56

^a Data represents the means ± SD of the IC₅₀ (µg/mL) of L. marginatus MeOH extract against tumor and normal cell lines. The SI indicates the IC₅₀ of VERO cells divided by that of B16F10 cells.

In addition, we evaluated the effect of L. marginatus MeOH extract in combination with minoxidil or bergamot oil (Fig. 2). We observed that L. marginatus MeOH extract did not affect the toxicity of minoxidil against B16F10 and VERO cells at any evaluated concentrations (Fig. 2a). Furthermore, the combination of 500 mg/mL L. marginatus extract + 6% bergamot oil significantly (p < 0.05) increased their individual growth inhibition effect on tumor and normal cell lines (Fig. 2b), whereas 62.5 mg/mL L. marginatus extract + 25% bergamot oil significantly (p < 0.05) increased their individual toxic effect against B16F10 cells, without affecting normal cells (Fig. 2b).

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Fig. 2.

Cell growth inhibition by (a) the combination of L. marginatus MeOH extract and (b) bergamot oil against B16F10 and VERO cell lines. Data represents the means ± SD of the percentage of growth inhibition at different treatment concentrations.

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3.2 In vivo hair growth promotion assay

Treatments shown in Table 1 were topically and daily administered to mice until day 21. Their body weights were recorded on days 0, 7, 14, and 21, resulting in no significant differences, as compared with the control group (untreated mice). However, significant alterations in body weights were found on days 7, 14, and 21, as compared with those on day 0 (Fig. 3). It was observed that on day 7, the weight significantly (p < 0.01) decreased (20.8%) after vehicle treatment. On day 14, mice treated with 5% minoxidil, vehicle, and the combination of 250 µg/mL L. marginatus extract and 50% bergamot oil showed a significant (p < 0.05) weight gain (12.4%, 15.6%, and 20.5%, respectively). On day 21, a significant (p < 0.01) weight increase was observed in all groups of treated mice, where the highest increase (34.3%) was observed after 500 µg/mL L. marginatus treatment. Body weights were not significantly altered in the untreated group control throughout the experiment.

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Fig. 3.

Mice body weights at days 0, 7, 14, and 21 following treatments with minoxidil, L. marginatus (Lm), bergamot oil, Lm + bergamot oil, Lm + minoxidil, and vehicle. Untreated mice were used as a negative control. Data represents the mean ± SEM of triplicate experiments. *P < 0.05, **p < 0.01, compared with results at day 0.

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To determine if the treatments promoted hair growth in BALB/c mice, hair abundance, length, and % regrowth were recorded on days 0, 7, 14, and 21. In addition, we photographed treatment results throughout the experiments to illustrate the effects of topical applications (Fig. 4).

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Fig. 4.

Mice hair growth at days 0, 7, 14, and 21 following treatments with vehicle, 5% minoxidil, 500 µg/mL Lm, 100% bergamot oil, 250 µg/mL Lm + 50% bergamot oil, and 250 µg/mL Lm + 2.5% minoxidil, compared with untreated control.

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Regarding hair abundance, we did not find significant differences in the treatments, as compared with the control group. However, significant (p < 0.01) differences were observed when results were compared with those on day 0 (Fig. 5). On day 7, the group treated with 5% minoxidil showed the highest (p < 0.01) increase in hair abundance with a value of 3 ± 0.5 (50% to 75%). On days 14 and 21, all groups presented significant (p < 0.01) differences, where maximum values on day 14 of 3.6 ± 0.3 were obtained in the group of mice treated with 500 µg/mL Lm and 250 µg/mL Lm + 2.5% minoxidil. Furthermore, on day 21, the vehicle and 250 µg/mL Lm + 2.5% minoxidil showed values of 4 ± 0 (100%).

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Fig. 5.

Mice hair abundance at days 7, 14, and 21 following treatments with minoxidil, Lm, bergamot oil, Lm + bergamot oil, Lm + minoxidil, and vehicle. Untreated mice were used as a negative control. Data represents the mean ± SEM of triplicate experiments. *P < 0.05, **p < 0.01, compared with results at day 0.

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Regarding mice hair length, we did not find significant differences between treatments and the control group. However, differences were observed as compared with results at day 0 (Fig. 6). On day 7, only 5% minoxidil significantly (p < 0.01) increased hair length with a value of 5 ± 1.2 mm. On days 14 and 21, hair length significantly (p < 0.01) increased in all treatments, as compared with results on day 0. The most significant (p < 0.01) results on day 14 were obtained after 250 µg/mL L. marginatus extract + 50% bergamot oil treatment with a value of 7.1 ± 0.7 mm and on day 21, after 250 µg/mL L. marginatus extract + 2.5% minoxidil treatment with a value of 8.8 ± 1.2 mm.

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Fig. 6.

Mice hair length at days 7, 14, and 21 following treatments with minoxidil, Lm, bergamot oil, Lm + bergamot oil, Lm + minoxidil, and vehicle. Untreated mice were used as a negative control. Data represent the mean ± SEM of triplicate experiments. *P < 0.05, **p < 0.01, compared with results at day 0.

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The percentage of hair regrowth was calculated by comparing treatment results with values obtained in the 5% minoxidil control group (Fig. 7), which is the standard therapy against alopecia. On day 7, it was found that the vehicle and Lm did not promote hair re-growth, whereas no significant differences were observed with the other treatments, as compared with the control group, which showed the highest percentages of re-growth with 49.7%. On days 14 and 21, no significant differences were observed between treatments and the control group. However, it was found that on day 14, hair re-growth was significantly (p < 0.05) higher in mice treated with bergamot oil (43.3%), Lm + bergamot oil (47.5%), and Lm + minoxidil (39.3%) than that of the control group (33%), whereas on day 21, all treatments induced significantly (p < 0.05) higher (> 20.4%) hair re-growth, as compared with the control group (17.3%). Lm + minoxidil caused the highest hair re-growth with 32.6%.

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Fig. 7.

Mice hair regrowth at days 7, 14, and 21 following treatments with minoxidil, Lm, bergamot oil, Lm + bergamot oil, Lm + minoxidil, and vehicle. Data represents the mean ± SEM of triplicate experiments. *P < 0.05, **p < 0.01, compared with the control group.

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4. Discussion

Alopecia is the loss of hair in any part of the body, occurring in approximately 2% of the human population (Villasante Fricke and Miteva, 2015). It may be related to distinct factors, including physiological and hormonal changes due to age, infectious diseases, eating disorders, pharmacological treatments, and cancer. Other factors such as depression, low self-esteem, stress, anxiety, and an inadequate quality of life may also be related to hair loss.

Some drugs are known to cause hair loss. They affect anagen follicles by suppressing mitosis in hair matrix cells (anagen effluvium) or by inducing their premature rest (telogen effluvium). Anagen effluvium is a common effect of antineoplastic agents, whereas telogen effluvium may be associated with anticoagulants, retinol (vitamin A), interferons, and antihyperlipidemic drugs. In contrast, hypertrichosis has been related to the use of cyclosporin, minoxidil, and diazoxide (Tosti et al., 1994).

Cancer is a multifactorial disease that is related to a pro-inflammatory process that generally affects hair follicles directly or indirectly, altering their growth or promoting hair loss. Pathophysiological disorders, such as the development of melanoma (skin cancer), also damage the follicles, which directly generate hair loss. On the other hand, pharmacological treatment for this disease may directly alter hair production, which causes hair loss in patients. Inflammatory reactions affect different mechanisms that promote hair growth, such as the process mediated by dihydrotestosterone (DHT), which regulates the hair growth stimulation cycle. Thus, its paracrine stimulation in the dermal follicles by androgens generates a regression in the irrigation of the bulb, which generates hypoxia and accumulation of oxidizing agents affecting the generation of new hair. Another effect related to DHT is stimulation of DKK-1 expression in dermal papilla cells, which inhibits the growth of keratinocytes of the external root sheath, which promotes hair loss.

Various natural and synthetic treatments for preventing hair loss and stimulating hair growth have been widely studied. However, there is no effective treatment that stimulates hair growth without causing adverse side effects. Minoxidil, one of the most widely used molecules for hair stimulation, has been highly studied. However, despite its high efficiency, it causes side effects, including headache, hair loss, and localized hypertrichosis, as observed in our study in the minoxidil-treated group, which was reduced by its combination with Lm (Fig. 4). Studies developed on healthy mouse cells (L929) showed cytotoxic and genotoxic effects at 2.0 mg/mL at 3 h of exposure. These results were observed in the present study at concentrations lower than 0.05%, inhibiting the growth of healthy cells (VERO) in more than 80% (Fig. 1b) (da Silva et al., 2023). VERO cells serve as a standard non-transformed control line for in vitro cytotoxicity assays, particularly in evaluating the effects of secondary metabolites from natural sources and in cancer research. (Hussein et al., 2020).

The use of natural compounds reduces the toxic effect on healthy cells, as observed in our in vitro results, since minoxidil showed significantly (p < 0.05) higher growth inhibition of tumor and normal cells, as compared with Lm and bergamot oil alone or in combination. Previous studies demonstrated the cytotoxic activity of Bergamot essential oil with an IC₅₀ of 110 µg/mL in colon cancer cells (HT-29). This is related to the antitumor activity of the oil in melanoma cells (B16F10), in which 40% inhibition of cell proliferation was observed at a concentration of 50% V/V (Fig. 1c). Recent studies showed that compounds such as bergamottin and 5-geranyloxy-7-methoxycoumarin resulted in respective IC₅₀ of 36.8 µM and 46.9 µM in human neuroblastoma cells (SH-SY5Y). The use of natural compounds did not affect non-tumor cells (VERO) after exposure for 48 h to concentrations of 10% to 50% V/V of bergamot oil (Fig. 1c). Furthermore, treatment of VERO cells with L. marginatus extract caused 60% growth inhibition at concentrations higher than 250 µg/mL (Fig. 1a). These results agree with a study showing that the in vitro use of a mixture of natural extracts (Chamomilla recutita, Urtica urens, Urtica dioica, Equisetum arvense, Achillea millefolium, and Ceratonia silique) showed lower cytotoxic effect in human keratinocytes (HaCaT) as compared with 1% minoxidil (Maugeri et al., 2021; Rossi et al., 2020; Türkoğlu et al., 2017). On the other hand, the antitumor effect (up to 70%) of LM against murine melanoma cells (B16F10) was at concentrations below 50 µg/mL, which increases the potential of using this extract to control melanoma (Fig. 1a), without affecting the growth of surrounding healthy cells, since it has an SI > 3 (Table 2). Murine melanoma is a cell line used in preclinical studies as a melanoma model (Potez et al., 2018).

The combination of natural treatments showed a similar effect in the different cells used (VERO and B16F10), maintaining a low inhibition index of growth (< 20%) in healthy cells at concentrations of 250 µg/mL Lm and 12.5% bergamotin oil and inhibiting melanoma cells above 20% at concentrations of 15.625 µg/mL Lm and 50% bergamotin oil. However, the anti-melanoma effect was lower, compared with the treatments alone (Fig. 2b). On the other hand, the combination of Lm with minoxidil was toxic for both cells in all combinations, which may be related to the cytotoxicity reported by this compound (Fig. 2a). These results differ with a study showing that the combination of 0.01 to 1 µM minoxidil and 0.01 to 1 nM retinoic acid promoted the proliferation of human dermal papilla cells and keratinocytes. Similarly, a reduction of the invasive potential of breast cancer cells (MDA-MB-468) was observed after combining 2.5 µM minoxidil with 2.5 µM ranolazine (Kwon et al., 2007; Qiu et al., 2022). These results are related to in silico studies using B-sitosterol, since an affinity with the DHT receptor was observed, which generates an anti-hair loss effect, whereas palmitic acid, acting as a ligand for PGD2, allows hair stimulation (Zamani et al., 2022).

The presence of three lipid molecules, including two phytosterols (B-sitosterol and lophenol) and a fatty acid (palmitic acid), isolated from the L. marginatus extract hexane fraction, has been previously reported (Gomez-Flores et al. 2019), which may be related to the results of our in vivo study, since mice treated with Lm alone or in combination with minoxidil or bergamot oil had a better state of their fur than that of the other treatments (Fig. 4). On the other hand, in the Serenoa repens (palmito) plant, families of similar metabolites such as fatty acids and phytosterols have been identified, suggesting a mechanism of action due to the blocking of the enzyme 5-alpha reductase and the decrease in the uptake of DHT by hair follicles. It also decreases the binding of DHT to androgenic receptors (Škulj et al. 2020). Similarly, the presence of palmitic acid in Cucurbita pepo and Ligustrum japonicum extracts, which have been used as treatments against alopecia, may be related to the inhibition of 5α reductase (Asnaashari and Javadzadeh 2020). Based on this, it is considered that the use of natural products with the presence of molecules that stimulate and prevent hair loss has a high potential for controlling alopecia generated by several factors, including cancer.

Previous studies have demonstrated that the topical use of minoxidil did not generate systemic conditions such as hypotension, tachycardia, and weight gain, which agrees with our results (Fig. 3). Furthermore, other studies showed that oral administration of minoxidil in some patients may generate fluid and salt retention, increasing patient’s weight (Suchonwanit et al., 2019). On the other hand, the development of hypertrichosis is one of the most frequent side effects of 5% minoxidil, which was observed in our study from day 7 (Fig. 4) (Panchaprateep et al., 2020; Suchonwanit et al., 2019).

Mice significant hair growth was observed from day 14, which agrees with reports that the hair growth phase occurs from this day onwards: However, other studies have shown that in mice exposed to minoxidil, their growth begins from the 3^rd week, and a significant hair growth was observed in all treatments alone and in combination, unlike other studies evidencing that hair growth increased until day 42, when treated with bergamot oil (Aldhalimi et al., 2014). Hair growth is an important aspect to consider since the factors for its loss may be related to distinct factors. Previous studies demonstrated that treatment for hair re-growth with testosterone or minoxidil in C57BL/6 mice showed exponential growth after day 14, as compared with the control group, which agrees with our study using the Balb/C mouse model (Fig. 7) for bergamot oil, minoxidil, and their combinations with Lm (Murata et al., 2013; Perna et al., 2019).

Finally, our results demonstrate that the hair growth promotion observed following exposure to the methanolic extract of Lophocereus marginatus may be related to, or interconnected with, various mechanisms. One such mechanism is the inhibition of the enzyme 5α-reductase, responsible for the conversion of testosterone into DHT, a key androgen involved in follicular miniaturization. This effect may be attributed to the presence of phytosterols such as β-sitosterol and lophenol, previously identified in the plant and known to modulate androgenic signaling.

Likewise, another possible mechanism could involve the blockade of androgen receptors, leading to the suppression of DKK-1, a Wnt pathway antagonist that negatively affects the proliferation of keratinocytes in the outer root sheath of the hair follicle. Therefore, the extract could help maintain the microenvironment necessary for a normal follicular cycle and effective hair regeneration.

On the other hand, prostanoid signaling pathways might also be modulated through the prostaglandin D2 (PGD2) axis, potentially due to the presence of palmitic acid, which has been proposed as a ligand for receptors involved in follicular regulation.

Taken together, these mechanisms may explain the in vivo stimulation of hair growth and support the potential of L. marginatus as a complementary agent in the treatment of alopecia. However, further molecular studies are required to confirm these hypotheses and clarify the specific targets involved.

5. Conclusion

L. marginatus MeOH extracts showed a significant potential for the development of hair treatments that enhance hair growth and prevent hair loss, as well as the control or elimination of skin cancer cells (melanoma).