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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1495-1499

Effects of standardized Cannabis sativa extract and ionizing radiation in melanoma cells in vitro


1 Department of Physiology, Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Radiology, Milad Hospital, Isfahan, Iran
3 Department of Pharmacology, Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission14-Dec-2016
Date of Decision30-Sep-2017
Date of Acceptance25-Feb-2018
Date of Web Publication27-Apr-2018

Correspondence Address:
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1394_16

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 > Abstract 


Context: Melanoma causes the highest number of skin cancer-related deaths worldwide. New treatment methods are essential for the management of this life-threatening disease.
Aims: In this study, we investigated the efficacy of a standardized Cannabis sativa extract alone or in combination with single radiation dose (6 Gy) in B16F10 mouse melanoma cells in an extract dose-dependent manner.
Materials and Methods: C. sativa extract at three concentrations (25, 12.5, and 6.25 μg/mL) alone for 72 h or in combination with radiation (24 h incubation after the extract treatment + 48 h incubation after exposure to radiation) were evaluated for cell viability of melanoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells were also treated with 6.25 μg/mL extract alone for 72 h before analyzing C. sativa-induced cell death by flow cytometry.
Results: Administration of the extract alone or alongside radiation substantially inhibited melanoma cell viability and proliferation in the extract dose response-dependent manner. The inhibition of melanoma cell viability was paralleled by an increase in necrosis but not apoptosis when melanoma cells were treated with the extract alone. Radiation alone did not have any antiproliferative effects, and radiation also did not synergize antiproliferative effects of the extract when the extract and radiation were combined.
Conclusion: Our data suggest that C. sativa extract may have significant health and physiological implications for the treatment of melanoma. The results of this study also indicate that B16F10 mouse melanoma cells are radioresistant. Taken together, these findings may lead to the identification of new therapeutic strategy for the management of melanoma.

Keywords: Anticancer effects, cancer treatment, cannabinoids, melanoma, radiation


How to cite this article:
Naderi J, Dana N, Javanmard SH, Amooheidari A, Yahay M, Vaseghi G. Effects of standardized Cannabis sativa extract and ionizing radiation in melanoma cells in vitro. J Can Res Ther 2020;16:1495-9

How to cite this URL:
Naderi J, Dana N, Javanmard SH, Amooheidari A, Yahay M, Vaseghi G. Effects of standardized Cannabis sativa extract and ionizing radiation in melanoma cells in vitro. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1495-9. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1495/231350




 > Introduction Top


“Cannabinoids” is a broad term covering a group of compounds extracted from the Cannabis sativa[1] and Cannabis indica plants. D9-tetrahydrocannabinol (THC) is the most prominent active component in this group of various compounds. THC is mainly responsible for the psychoactive effects of the plant.[2],[3] Cannabinoids exert a variety of effects through binding to specific receptors. At least two types of cannabinoid receptors (CBs) have been characterized as CB1 and CB2.[4],[5]

Many studies have reported the antiemetic activity of Cannabis extracts and cannabinoids on chemotherapy-associated emesis. [2,6-8] The results of preclinical trials have shown that THC possesses the antiemetic activity in animal models in radiation-induced emesis.[8],[9] Cannabis extracts and cannabinoids also have been useful in the pain management[10],[11] and treatment of inflammation.[11],[12] The antiproliferative effects of cannabinoids in a variety of cancer cell lines, including pancreatic, melanoma, glioma, lymphoma, lung, and breast, have been reported. [3,13-15] However, potential therapeutic interactions of Cannabis extracts and cannabinoids with conventional cancer therapies, such as radiation, are largely unknown. Exposure to radiation leads to either necrosis or apoptotic cell death. The ability of different agents to promote the cell death is of central importance when selecting specific anticancer treatment.[16] This study was performed to find whether C. sativa extract treatment alone or in combination with single radiation dose (6 Gy) may reduce the cell viability paralleled by increased cell death in mouse melanoma cells in vitro.

Melanoma is the third-most common skin cancer but the leading cause of skin cancer-related deaths.[17] The incidence of melanoma is increasing worldwide, and the prognosis for patients with advanced disease remains weak.[18] Treatment options for melanoma include surgical excision, chemotherapy, radiation therapy (RT), immunotherapy, targeted therapy, prevention of angiogenesis, and combination therapies.[19],[20],[21] Surgery is the initial treatment for primary and locoregional melanoma, but the other treatment options above are applied for metastatic setting.[20] Although melanoma has been known a radio-resistant tumor, emerging data have shown that RT is now recognized an effective therapy in some settings.[20] In addition to current treatments, medicinal plants are important for cancer treatment.[22],[23] Effects of medicinal plants in various cancer cell lines[24],[25] including melanoma cells have been studied.[26],[27] Although there are different treatment options for melanoma, any single treatment remains largely unsuccessful due to the poor duration of response, resistance, and side effects.[21] Therefore, the aim of this study is to examine the response of mouse melanoma cells in vitro following C. sativa extract treatment alone or in combination with radiation. Cell viability and induction of apoptosis/necrosis were assessed in this study.


 > Materials and Methods Top


Plant extraction

The plant was identified at the Botany Department of the Faculty of Sciences, Isfahan University, and a voucher specimen of the plant has been deposited in the herbarium of Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran. C. sativa dry flowers and leaves were extracted at room temperature with CO2 and standardized based on 4% cannabidiol, according to the information provided by the supplier company (Barij-Esans Co., Iran).

Reagents

Dulbecco's modified eagle medium (DMEM) media, fetal bovine serum (FBS), and Trypsin were from Gibco-BRL (UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Germany). Annexin V-FITC Apoptosis Detection Kit was obtained from eBioscience (Austria).

Cell culture

The B16F10 mouse melanoma cell line (obtained from Pasteur Institute) was cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. Cells were cultivated until 3–4 days to reach nearly 80% confluency. The extract of C. sativa was added to the cells once in DMEM/10% FBS. Untreated cells without the C. sativa extract were included for unstimulated control.

Radiation therapy

The single absorbed dose of ionizing radiation (IR) was 6 Gy from 6 MV photons at a source-to-surface distance of 100 cm. The IR was delivered by an RT machine (Siemens ONCOR linear accelerator) at room temperature.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Following almost 80% confluence, melanoma cells were plated into two 96-well plates (3000 cells/well) and allowed to adhere overnight. The cells, in one plate, were then pretreated for 24 h with varying doses of C. sativa extract (25, 12.5, and 6.25 μg/mL), before being subjected to the single radiation dose (6 Gy). The cells, in this plate, were incubated for 48 h following exposure to IR. The cells, in another plate, only were treated for 72 h with the same doses of the extract as the first plate, without IR. Following these procedures, MTT was added to these two plates according to the instruction of the supplier company and incubated for 4 h. The absorbance was measured by a microplate reader (BioTek, OD 570 nm with 630 nm correction). The experiments were performed in series of 3 wells per treatment and repeated three times.

Flow cytometry

Following almost 80% confluence, cells were seeded into 24-well plates (1,00,000 cells/well) and left to adhere overnight. Cells were treated with the lowest effective dose of the extract (6.25 μg/mL) for 72 h. Following 72 h of treatment, cells were washed in PBS followed by resuspending cells in Binding Buffer. Annexin V-FITC was added to the cell suspension and incubated for 10 min at room temperature. Propidium iodide (PI) was added to cell suspension according to the information provided by the supplier company. Samples were analyzed using a BD FACSCalibur flow cytometer in triplicate in one independent biological replicate.

Statistical analysis

All data are shown as mean ± standard deviation. Differences between variable and untreated control groups were determined by appropriate ANOVA followed by Tukey–Kramer test when appropriate. Statistical analysis was performed using SPSS Statistics version 21. P = 0.05 was considered statistically significant.


 > Results Top


C. sativa extract reduces the cell viability in B16F10 melanoma cells

We performed MTT assays to determine if the C. sativa extract alone or alongside radiation would affect cell metabolic activity as decreased metabolic activity may represent inhibition of cell proliferation or decreased cell viability. Actively proliferating melanoma cells were treated with three doses of C. sativa extract (25, 12.5, and 6.25 μg/mL) alone for 72 h or in combination with radiation (24 h incubation after the extract treatment + 48 h incubation after exposure to radiation). MTT was then added and metabolized for 4 h. 25, 12.5, and 6.25 μg/mL C. sativa extract treatments significantly decreased the cell metabolic activity by 93.45%, 91.73%, and 87.76%, respectively relative to untreated cells (n = 3; P = 0.05) [Figure 1]. 25, 12.5, and 6.25 μg/mL C. sativa extract treatments in combination with radiation significantly decreased the cell metabolic activity by 92.69%, 91.38%, and 86.62%, respectively, relative to untreated cells (nonradiation untreated cells) (n = 3; P = 0.05) [Figure 1]. There is no significant difference between the metabolic activity of treated cells by C. sativa extract and C. sativa extract-treated cells + radiation [Figure 1]. Therefore, the combination treatment of C. sativa extract + radiation did not produce a synergistic reduction of the metabolic activity. Radiation alone also did not have any significant effects on the metabolic activity of melanoma cells.
Figure 1: Assessment of the metabolic activity in response to the Cannabis sativa extract alone or in combination with radiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. B16F10 cells were exposed to three doses of the extract alone for 72 h or in combination with 6 Gy radiation (24 h incubation after the extract treatment + 48 h incubation after exposure to radiation). Data reflect mean ± standard deviation where untreated control is set to 100% (n = 3). *P ≤ 0.05 versus untreated control

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Extract of C. sativa induces cell death

To determine the possibility that cell death might have contributed to the reduction in the metabolic activity and cell viability detected, the induction of apoptosis and/or necrosis was analyzed by annexin V-FITC and PI. We did not examine the effect of radiation alongside the C. sativa extract in melanoma cells since the combination treatment of the C. sativa extract + radiation did not produce a synergistic reduction in the cell viability, and radiation alone also did not have any significant effects on the cell viability of melanoma cells. The dose of 6.25 μg/mL C. sativa extract was selected to investigate cell death as this dose has been the lowest effective dose of the extract in this study, which has been evaluated, and this dose reduced the cell viability less than other doses (25 and 12.5 μg/mL). Therefore, cells were treated with 6.25 μg/mL extract for 72 h, stained with annexin V and PI before analyzing C. sativa-induced cell death by flow cytometry. The melanoma cells responded to the extract treatment as the percentage of healthy, necrotic, and apoptotic cells was 79.66%, 20.04%, and 0.30%, respectively, compared with untreated cells that the percentage of their healthy, necrotic, and apoptotic cells was 98.48%, 0.56%, and 0.96%, respectively (n = 1) [Table 1]. Necrosis was clearly detected in response to the extract treatment according to our results. Taken together, this finding corresponds to the data from the MTT assay, supporting that C. sativa extract promotes cell death in mouse melanoma cells.
Table 1: Effects of the Cannabis sativa extract (6.25 μg/ml) on cell death in B16F10 melanoma cells following 72 of treatment as shown by flow cytometry

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 > Discussion Top


This study shows that B16F10 mouse melanoma cells respond to C. sativa extract alone or alongside radiation (6 Gy) by reducing the cell viability in a C. sativa extract dose-dependent manner. However, our in vitro data suggest that the mouse melanoma cells are radioresistant. Flow cytometry data show that C. sativa extract at 6.25 μg/mL for 72 h significantly induces necrosis. This result supports the data from MTT assay, which C. sativa extract reduces the cell viability paralleled by an increase in the cell death.

Both marijuana tar extract and synthetic THC were found to hinder apoptosis while stimulating necrosis.[28] Cannabis leaves have been shown to promote necrotic cell death by secreting THA acid (THCA) into leaf tissues. THCA treatment also promotes necrotic cell death in plants which do not produce cannabinoids.[29] Our results are in agreement with the results specified above. We observed that C. sativa extract at 6.25 μg/mL for 72 h significantly induces necrotic cell death while inhibiting apoptosis in melanoma cells. Further study is required to find whether the active components in C. sativa or lower doses of C. sativa extract may induce apoptosis in melanoma cells in vitro. It is important to highlight that THC is not cytotoxic at concentrations <5 μg/mL in A549 lung tumor cells, but induces necrosis at higher levels with a lethal concentration 50 = 16–18 μg/mL.[30] The study demonstrated that C. sativa extract is cytotoxic at three concentrations (6.25, 12.5, and 25 μg/mL), investigated in the present study, and the extract at 6.25 μg/mL also induces necrosis. This inconsistency in our results with results of Sarafian et al.[30] may arise from using C. sativa extract instead of THC, different cell types (B16F10 melanoma cells and A549 lung tumor cells), and incubation time. It is noteworthy that cannabinoids, primarily THC, have demonstrated psychotropic effects.[31] Hence, further study is needed to examine whether lower doses of the C. sativa extract can reduce cytotoxicity and psychotropic effects of the extract in vitro and in vivo.

Several studies support a role for Cannabis extracts and cannabinoids in growth inhibition of tumor cells or tumor cell death.[32],[33],[34],[35] Our results are consistent with aforementioned studies and show that C. sativa extract significantly reduces cell viability of melanoma cells and induces cell death. Further investigation is required to find whether these effects of C. sativa extract are selective and whether C. sativa extract may have antitumor effects in vivo as well.

Although melanoma has historically been considered a relative radio-resistant tumor, RT has been recommended in a number of clinical settings including definitive, adjuvant, and palliative management of melanoma.[20],[36],[37] However, our data support that B16F10 mouse melanoma cells are radioresistant. Our finding is consistent with at least three studies[38],[39],[40] that showed melanoma is intrinsically radio-resistant in vitro. On the other hand, the response of melanoma to radiation has been shown to depend on tumor volume, radiation dose, and fraction size.[41] In the present study, single-radiation dose was 6 Gy while early in vitro studies of melanoma radiosensitivity by clonogenic assays indicated that successful treatment required high dose per fraction RT.[42],[43] Theoretically, the ability of melanoma cell to conquer sublethal DNA injuries induced by radiation proposes that, clinically, melanoma should be more sensitive to large doses per fraction than to lower fraction doses.[41] This hypothesis is in contrast to at least two randomized clinical trials, showing similar rates of response with low–to-medium dose fractions (2.5–5 Gy) and larger dose fractions (8–9 Gy). Moreover, the clinical trial showing an improvement in regional disease control using low dose per fraction (2.4 Gy) RT, further dispelling the myth that RT is only effective for melanoma if given with high doses per fraction.[43] Although the study of Mahadevan et al.[20] proposes that the fractionation in melanoma is controversial, the evidence suggests that clinicians should be comfortable with using low-dose-per-fraction RT in proper settings.[43] It should be taken into consideration that the rate of cell viability for melanoma cells exposed to single radiation doses in vitro was found to differ markedly among individual cell lines.[42],[43] Thus, melanoma is a tumor type that shows considerable variation in radioresponsiveness.[42],[44],[45] Thus, optimal RT of melanoma will probably requires a personalized treatment approach. In vitro experiments for prediction of radiocurability, the optimum utilization of RT, and choice of treatment option for individual melanoma patient appear therefore clearly warranted.[42]

The single doses of gamma rays and protons in the range from 8 to 24 Gy following 7 days after irradiation significantly reduced estimated cell viability in HTB140 human melanoma cells, as compared to nonirradiated control.[16] Although it was initially presumed that apoptosis ensued relatively fast (within hours) after radiation, the evidence demonstrates that apoptosis can ensue at long times after radiation (out to 20 days) in some cell types.[46] Our results may confirm the aforementioned studies as in the present study melanoma cells did not respond to radiation following 48 h postradiation incubation. Further studies are needed to find whether melanoma cells may respond to radiation following longer postradiation incubation time.

The effect of adjuvant RT in the treatment of melanoma remains controversial and is underused.[37],[47] Our study is the first research that investigated the combined adjuvant cellular effects of C. sativa extract and radiation in mouse melanoma cells in vitro. This in vitro study demonstrates that radiation (6 Gy) does not have any synergistic antitumor effects in melanoma when combined with C. sativa extract in an extract dose-dependent manner.


 > Conclusion Top


This study provides the first evidence of antitumor effects of C. sativa extract, when administered alone or in combination with radiation, to mouse melanoma cells in vitro. Our results may verify the value of C. sativa extract for the treatment of melanoma and may complement the therapeutic profile of C. sativa extracts administration in the future. Precise mechanisms and the biologic basis of C. sativa extract-induced cell death need to be explored with well-designed studies. Further studies are required to evaluate antitumor effects of C. sativa in vivo and also to investigate in vitro cellular effects of combined C. sativa extract and radiation in different single and fractionated doses of radiation, and longer postradiation incubation time.

Acknowledgment

We thank Dr. Abed, Barij-Esans Co., for kindly providing the extract.

Financial support and sponsorship

This study was funded by Isfahan University of Medical Sciences (grant number 293212).

Conflicts of interest

There are no conflicts of interest.



 
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