Effects of dichloromethane and N-butanol fractions of Nigella sativa on ACHN and GP-293 cell line morphology, viability, and apoptosis
Samira Shahraki1, Sara Hosseinian2, Elham Shahraki3, Mehdi Kheirandish4, Abolfazl Khajavirad2
1 Department of Physiology, Zahedan University of Medical Sciences, Zahedan; Department of Physiology, Mashhad University of Medical Sciences, Mashhad, Iran
2 Department of Physiology, Mashhad University of Medical Sciences; Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
3 Department of Nephrology, Internal Medicine, Ali Ibne Abitaleb Hospital, Zahedan University of Medical Sciences, Zahedan, Iran
4 Department of Physiology, Zahedan University of Medical Sciences, Zahedan, Iran
|Date of Submission||19-Nov-2022|
|Date of Acceptance||13-Jun-2023|
|Date of Web Publication||27-Jul-2023|
Dr. Abolfazl Khajavirad
Applied Biomedical Research Center, Mashhad University of Medical Sciences; Department of Physiology, Faculty of Medicine, Azadi Square, Mashhad, I. R. Iran
Source of Support: None, Conflict of Interest: None
Background: Renal cell carcinoma (RCC) is among the top death-causing cancers. Medicinal herbs can also have beneficial effects on RCC treatment. In this project, we aimed to study the antitumor effect of dichloromethane and N-butanol fractions of hydroalcoholic extract of Nigella sativa (N. sativa) on the morphology, viability, and apoptosis of ACHN (human renal adenocarcinoma) and GP-293 (normal renal epithelial) cell lines.
Materials and Methods: In this experimental study, N-butanol and dichloromethane fractions of N. sativa were obtained, and ACHN and GP293 cell lines were treated with various concentrations of dichloromethane (0–100 μg/mL) and N-butanol (0–12.5 μg/mL) fractions for 24, 48, and 72 hours. Then, morphological changes, viability, and apoptosis were investigated.
Results: Our results indicated that dichloromethane and N-butanol fractions cause morphological changes and significant decreases in the percentage of live cells in the ACHN cell line, in a dose- and time-dependent manner. In the GP-293 cell line, however, a lower toxicity was observed in comparison with that found for ACHN. The results of flow cytometry showed an apoptotic effect of dichloromethane and N-butanol fractions on the ACHN cell line but a higher rate of apoptosis induction for the total extract compared to the two fractions in the renal cancer cell line compared to the normal cell line.
Conclusion: Our findings demonstrated that these two fractions of N. sativa induce inhibitory effects on the ACHN cell line morphology and viability. These effects were lower than those induced by the total extract. In addition, the two fractions caused more marked effects in the renal cancer cell line compared with the GP-293 cell line.
Keywords: Flow cytometry, fractions of extract, MTT, Nigella sativa, renal cell carcinoma
|How to cite this article:|
Shahraki S, Hosseinian S, Shahraki E, Kheirandish M, Khajavirad A. Effects of dichloromethane and N-butanol fractions of Nigella sativa on ACHN and GP-293 cell line morphology, viability, and apoptosis. Adv Biomed Res 2023;12:200
|How to cite this URL:|
Shahraki S, Hosseinian S, Shahraki E, Kheirandish M, Khajavirad A. Effects of dichloromethane and N-butanol fractions of Nigella sativa on ACHN and GP-293 cell line morphology, viability, and apoptosis. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:200. Available from: https://www.advbiores.net/text.asp?2023/12/1/200/382400
| Introduction|| |
The most deadly form of genitourinary tract cancer is renal cell carcinoma (RCC). RCC includes a sort of pathologically distinguished tumor subtype of the kidney. Clear cell RCC is the most prevalent type of RCC. The incidence of RCC in men is lower than in women. Smoking and obesity are the most prevalent causes of this disease. Also, the risk of developing RCC in diabetic patients is high. The classic triad of RCC symptoms are pain of flank, clear abdominal mass, and hematuria. Surgery, hormone therapy, immunotherapy, and chemotherapy are therapeutic strategies employed for RCC management.
The use of medicinal plants has a special place in folklore and traditional medicine. Particularly, medicinal plants are generally regarded as agents with less side effects and they have lower costs than conventional treatments. Medicinal plants are precious resources of new drugs, even those used for the treatment of malignancies. They are not only cheaper and more readily available, but also better tolerated by patients. Medicinal herbs can also have beneficial effects in RCC treatment., Nowadays, medicinal plants were utilized for treating several diseases such as infections, asthma, and gastrointestinal and cardiovascular diseases. One of these herbs is Nigella sativa (N. sativa), which is a plant of the Dicotyledonae family. N. sativa is also popular as black cumin, black caraway seed, Kalonji, Hak Jung Chou, and Panacea. The seeds of this plant contain 36–38% nonvolatile oil and 0.4–2.5% essential oils, alkaloids, and saponins. Appreciable quantities of unsaturated fatty acids, mainly polyunsaturated fatty acids, are in the fixed oil of N. sativa, which form the bulk of oil ranging from 48 to 70%, while monounsaturated (18–29%) and saturated fatty acids (12–25%) are in lesser ratios. The essential oils of these plants include thymoquinone (TQ) (27.8–57.0%), p-cymene (7.1–15.5%), t-anethole (0.25–2.3%), 4-terpineol (2.0–6.6%), longifolene (1.0–8.0%), and carvacrol (5.8–11.6%). N. sativa has promising pharmacologic and therapeutic properties such as antioxidant, anticancer and anti-mutation, anti-hepatic and anti-nephrotoxicity, antidiabetic, anti-inflammatory, antiulcer, analgesic, and sedative effects.
TQ as the main component of N. sativa's volatile oil, as well as dithymoquinone, has shown beneficial effects in the treatment of cancer. Many studies documented the anticancer and antitumor activities of TQ on cervical, breast, colon, lung, larynx, skin, and prostate cancers.
In our project, the effects of dichloromethane and N-butanol fractions of hydroalcoholic extract of N. sativa on the morphology, viability, and apoptosis of cells were evaluated in human kidney adenocarcinoma cell line (ACHN) and normal human kidney epithelial cell line (GP-293); also, results were compared with those obtained using N. sativa total extract in our previous study.
| Materials and Methods|| |
In this experimental study, fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purveyed from Gibco (Grand Island, USA), Annexin V and propidium iodide (PI) kit was purchased by BioVision (USA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) was supplied from Sigma (St Louis, MO, USA).
ACHN (human renal adenocarcinoma) and GP-293 (normal renal epithelial) cell lines were prepared from Pasteur Institute (Tehran, Iran). The cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin or streptomycin.
N. sativa hydroalcoholic extract was prepared as previously described. To make the fractions, 20 g of hydroalcoholic extract of N. sativa was mixed with 200 ml of ethanol and transferred to a decanter funnel. Successively, the concentrated extract was fractionated using equal volumes of n-hexane, dichloromethane, ethyl acetate (EtOAc), and N-butanol (n-BuOH), respectively, three times. In each step, the solvent was added to the funnel, and all the fractions were extracted. In the end, isolated fractions were concentrated at 50°C under reduced pressure to dryness. The dichloromethane and N-butanol fractions were subjected to cytotoxic and apoptosis assays.
The viability of cells was assessed with the MTT test. The MTT assay is a colorimetric method developed based on the reduction and breakdown of yellow tetrazolium crystals. Cells in the exponential stage of growth were harvested by trypsinization and centrifugation. After performing the trypan blue test, 5 × 103 ACHN cells and 1 × 104 GP-293 cells were seeded in each well of the 96-well plates and allowed to grow for 24 hours. Each cell line was incubated for 24, 48, and 72 hours with different concentrations of dichloromethane and N-butanol fractions of N. sativa (which were equal to 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, and 2000 μg/ml concentrations of total extract). ACHN and GP-293 cell lines were treated with each concentration of dichloromethane and N-butanol fractions of N. sativa in triplicates. After 24, 48, and 72 hours of incubation, MTT solvent was added to each well. After 4-h incubation, the medium was removed and 200 ml of dimethyl sulfoxide (DMSO) was added to each well. Finally, the absorption was measured at 570 nm (with 620 nm used as a reference) by an ELISA reader. “The results that have not reported here show that DMSO has a cytotoxic effect at ≥ 1% concentrations in these cell lines. Therefore, we used DMSO at 0.5% concentration for total extract and dichloromethane and N-butanol fractions of N. sativa.”
Following 24-, 48-, and 72-h treatments with various concentrations of the two fractions of N. sativa, cells were investigated under an inverted light microscope. The control group in this examination is untreated cells.
Apoptosis and necrosis quantification
To measure apoptosis in the ACHN and GP-293 cell lines, we used the BioVision Kit. There are Annexin V and PI in this kit. Both ACHN and GP-293 cell lines were treated with dichloromethane and N-butanol fractions of hydroalcoholic extract of N. sativa for 48 hours. Then, the cells were collected, treated with 500 ml of binding buffer, and incubated for 5 minutes in the dark at room temperature. In the next step, cells were incubated with 5 ml of Annexin V and 5 ml of PI, for 10 minutes under the same conditions. The cells were then assessed with a flow cytometry machine. Each well of treated cells was placed in separate flow cytometry plots, and the primary or secondary apoptosis or necrosis cells were separated.
For normally distributed variables, a one-way analysis of variance (ANOVA) was used. When a significant difference was seen, the post hoc Tukey test was performed for comparing the results between the experimental groups. The results are shown as mean ± SEM. P <0.05 was considered statistically significant.
| Results|| |
Untreated ACHN and GP-293 cells (i.e., control) had a fibroblast-like appearance and were spindle-shaped as observed by an inverted light microscopy [Figure 1]a and [Figure 1]b, [Figure 1]i and [Figure 1]j.
|Figure 1: Morphological changes in GP-293 and ACHN cell lines after 48 and 72 h at 1000 μg/ml: control GP-293 cells at 48 h (a), control ACHN cells at 48 h (b), GP-293 cells treated with total extract of Nigella sativa at 48 h (c), ACHN cells treated with total extract of Nigella sativa at 48 h (d), GP-293 cells treated with dichloromethane fraction of N. sativa at 48 h (e), ACHN cells treated with dichloromethane fraction of N. sativa at 48 h (f), GP-293 cells treated with N-butanol fraction of N. sativa at 48 h (g), and ACHN cells treated with N-butanol fraction of N. sativa at 48 h (h), control GP-293 cells at 72 h (i), control ACHN cells at 72 h (j), GP-293 cells treated with total extract of Nigella sativa at 72 h (k), ACHN cells treated with total extract of Nigella sativa at 72 h (l), GP-293 cells treated with dichloromethane fraction of N. sativa at 72 h (m), ACHN cells treated with dichloromethane fraction of N. sativa at 72 h (n), GP-293 cells treated with N-butanol fraction of N. sativa at 72 h (o), and ACHN cells treated with N-butanol fraction of N. sativa at 72 h (p)|
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We previously showed the morphological changes caused by the total extract of N. sativa in ACHN and GP-293 cell lines [Figure 1]c and [Figure 1]d, [Figure 1]k and [Figure 1]l. In normal renal epithelial cell line, 24 hours after treatment with the dichloromethane fraction of 50–1500 μg/ml, no morphologic changes were observed, but higher cell numbers were found as compared to control wells. In cells treated with dichloromethane fractions of 1750 and 2000 mg/ml, the living cells' number and density of cell were reduced compared with the control group. By increasing the incubation period, morphological alterations were caused at lower doses and with higher intensity [Figure 1]e and [Figure 1]m. Following 24-h treatment with the dichloromethane fraction of 50–500 μg/ml, ACHN cells were attached to the well bottom, but some of the cells were granulated. Cell incubated with 750–2000 μg/ml dichloromethane fractions had a lower density of cell compared with control wells and lost their spindle-like shape. Increasing incubation time caused morphological changes with greater intensity [Figure 1]f and [Figure 1]n.
Treatment of the GP-293 cell line with N-butanol fraction of 50–1750 μg/ml for 24 hours produced no changes, whereas 2000 μg/ml concentration reduced the number of live cells compared with the controls. 48 and 72 hours after treatment, the morphological changes were seen at lower doses of the fraction and they were of greater intensity [Figure 1]g and [Figure 1]o. A morphological study of ACHN cell line incubated with the N-butanol fraction of 50–1000 μg/ml for 24 hours showed multiple colonies all around the well. Wells incubated with 1250–2000 μg/ml of the N-butanol fraction had lower cell density and cells were isolated from the well bottom, compared with controls. With increasing time and concentration, morphological changes were observed with greater intensity [Figure 1]h and [Figure 1]p. As shown in [Figure 1], the morphological findings of the current study were compared with the previous study.
Our previous study showed that N. sativa total extract is able to decrease cell viability in ACHN and GP-293 cell lines. However, [Figure 2] displays the growth inhibitory effect of the total hydroalcoholic extract in the GP-293 and ACHN cell lines presented in our previous article.
|Figure 2: Growth inhibition of GP-293 and ACHN cell lines with N. sativa total extract after 24, 48, and 72 h. Results are mean ± SEM (n = 3). P <0.05*, P < 0.01**, and P < 0.001*** compared to control|
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In GP-293 cells, after 24-h incubation with 2000 μg/ml of dichloromethane fraction cell viability significantly decreased compared with the control wells (P < 0.001). Additionally, treatment of GP-293 cells with dichloromethane fraction of 1750 μg/ml for 72 and 48 hours caused a significant decrease in cell viability compared with the control group (P < 0.01) [Figure 3]. In the ACHN cell line, after 24-h treatment 50 μg/ml of dichloromethane nonsignificantly decreased cell viability compared with the control group, but at 1000 μg/ml concentration, dichloromethane fraction significantly reduced cell viability (P < 0.01). With increasing time, a significant reduction in the ACHN cell viability was seen following treatment with 50 μg/ml of dichloromethane fraction compared with the control cells (P < 0.001) [Figure 3]. Also, half maximal inhibitory concentration (IC50) values of dichloromethane fraction, in the ACHN cell line, were 5637, 3365, and 2855 μg/ml, and in the GP-293 cell line, they were 48877, 37784, and 17564 μg/ml for 24-, 48-, and 72-h treatments, respectively.
|Figure 3: Growth inhibition of GP-293 and ACHN cell lines with dichloromethane fraction of N. sativa after 24, 48, and 72 h. Results are mean ± SEM (n = 3). P <0.05*, P < 0.01**, and P < 0.001*** compared to control|
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24 h after incubation with 2000 μg/ml of N-butanol fraction, a nonsignificant reduction in the GP-293 viability of cells was observed in comparison with controls. Also, 48 hours after incubation, a nonsignificant reduction in cell viability was only seen for 1750 and 2000 μg/ml concentrations. After 72-h treatment with 1250 μg/ml of N-butanol fraction, a nonsignificant decrease in GP-293 cell viability was seen compared with the control group [Figure 4]. 24 hours after treatment, ACHN cell line with N-butanol fraction at 250 μg/ml concentration nonsignificantly reduced cell viability, but this fraction at concentrations ≥750 μg/ml caused significant reductions in cell viability as compared to the control group (P < 0.001). After 48 and 72 hours of incubation with ≥100 μg/ml of N-butanol fraction, ACHN cell viability significantly reduced in comparison with controls (P < 0.001) [Figure 4]. Furthermore, IC50 values of N-butanol fractions in the ACHN cell line at different time points were 11393, 5766, and 5410 μg/ml, and in the GP-293 cell line, they were 210876, 209897, and 40096 μg/ml for 24-, 48-, and 72-h treatments, respectively.
|Figure 4: Growth inhibition of GP-293 and ACHN cell lines with N-butanol fraction of N. sativa after 24, 48, and 72 h. Results are mean ± SEM (n = 3). P <0.05*, P < 0.01**, and P < 0.001*** compared to control|
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The apoptotic action of total extract on ACHN and GP-293 cell lines is shown in [Figure 5]. These data were presented in our previous article.
|Figure 5: Apoptotic effects of total extract, dichloromethane, and N-butanol fraction of N. sativa on GP-293 and ACHN cell lines after 48 h. Results are mean ± SEM (n = 3). P <0.001*** compared to control|
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Following 48-h treatment with different concentrations of dichloromethane and N-butanol fractions, no significant effect with respect to apoptosis rate was found in the GP-293 or ACHN cell line compared with the control [Figure 5].
| Discussion|| |
In our research, the effect of dichloromethane and N-butanol fractions of N. sativa on GP-293 and ACHN cell lines was evaluated; also, we compared the data obtained in the present work with those that we previously reported for the total extract of the plant. The results showed that both fractions in a time- and dose-dependent way cause morphological changes and decrease cell viability in both cell lines. However, this effect was more marked in the renal cancer cell line than in the GP-293 cells. The sixth most common reason for the death of cancer in men and the 10th in women is kidney cancer. Signaling pathways that are involved in kidney cancer are angiogenic, Phosphatidylinositol-3-kinase or alpha serine/threonine-protein kinase or The mammalian target of rapamycin, Wntß-catenin, and hepatocyte growth factor or mesenchymal-epithelial transition factor pathways. Anticancer properties of N. sativa including induction of morphological changes and apoptosis, as well as reduction in cell viability, were previously shown against kidney cancer. Furthermore, N. sativa is used for the cure of several cancers, for instance, leukemia, as well as skin, hepatic, breast, cervical, and prostate cancers. To determine which N. sativa components have anticancer effects, various fractions of the plant were studied. Basically, different fractions or groups of compounds with similar chemophysical characteristics are extracted from plants to divide them into multiple chemical groups. For this purpose, the total extract of the plant was fractionated using the following solvents, respectively, n-hexane, dichloromethane, ethyl acetate, and N-butanol solution. In the current work, the effects of dichloromethane and N-butanol fractions were evaluated on morphology, viability, and apoptosis of ACHN and GP-293 cell lines.
The results of our study showed that dichloromethane and N-butanol fractions produced more marked morphological changes in the ACHN cell line, in a dose- and time-dependent way, compared with the effects observed in GP-293 cells. However, the N-butanol fraction was less toxic for normal renal cells than the dichloromethane fraction. It may be concluded that the N-butanol fraction is better tolerated by normal cells than the dichloromethane fraction. Furthermore, our previous study indicated that the total extract of N. sativa induced morphologic changes in the ACHN cell line that were more severe than those observed in the GP-293 cell line. Moreover, morphological changes caused by the total extract of N. sativa were more evident than its two fractions, dichloromethane and N-butanol.
Also, the viability of ACHN cell lines decreased with N-butanol fraction in a dose- and time-dependent way, while it produced low toxicity in GP-293. The potency and the selectiveness of the effect of this fraction on the inhibition of ACHN cell line growth were similar to those of dichloromethane. In our previous study, we showed similar action for N. sativa ethyl acetate fraction in kidney cancer cells. Furthermore, N. sativa total extract was more toxic effect toward the ACHN cell line than on the GP-293 cells. Therefore, these results are in line with previous articles, which also showed the viability-reducing effect of N. sativa extract in cancer cell lines., Mbarek et al. showed that the N-butanol fraction of N. sativa extract produced higher toxicity in the ICO1 cell line (sheep heart carcinoma) than VERO (monkey kidney carcinoma) and BSR (hamster kidney carcinoma) cell lines. Consistently, in the current study, we showed a stronger and more selective effect of N-butanol fraction against the ACHN cell line. It is suggested that the anticancer effect of N. sativa might be attributed to its polar compounds. In this regard, the N-butanol fraction contains polar compounds, while the dichloromethane fraction has non-polar or semi-polar compounds including polyphenols, fatty acid, and lipids., Accordingly, it is postulated that polar compounds of N-butanol fraction may improve their anticancer action.
The flow cytometry analysis demonstrated that the mean percentage of apoptosis caused by N-butanol fraction in ACHN and GP-293 cell lines was “%9.95” and “%1.24” and that induced by dichloromethane fraction was “%14.1” and “%0.5,” respectively. In addition, dichloromethane fraction at lower concentrations increased ACHN cell line apoptosis, but at higher concentrations, it reduced kidney cancer cell apoptosis. It may be suggested that at higher doses, the dichloromethane fraction effect in the ACHN cell line shifts from inducing apoptosis to necrosis. Based on the results of this investigation, apoptosis induction by dichloromethane fraction in the ACHN cell line was higher than that caused by N-butanol fraction. Several pieces of evidence showed that different fractions of N. sativa, depending on their constituents, have different effects on apoptosis against variant cell lines., According to previous studies, the total extract of N. sativa is able to induce apoptosis in hepatic and renal cancer cell lines. In this line, our previous study indicated that the total extract and ethyl acetate and n-hexane fractions of N. sativa produce higher apoptosis in ACHN cells than in GP-293 cells. Our results also indicated that the apoptotic effect of dichloromethane and N-butanol fractions in the GP-293 and ACHN cell lines was not significantly different from the untreated cells (control group). Consistently, our previous study demonstrated that the apoptosis rate in cells treated with two fractions of N. sativa includes ethyl acetate and n-hexane was not significantly different from that observed in the untreated cells. In addition, analysis of apoptotic effects of dichloromethane and N-butanol fractions in human renal adenocarcinoma and normal renal epithelial cell lines showed that the eminent cytotoxicity mechanism for both of these fractions might be suggested apoptosis. The possible mechanisms via which apoptosis was induced in these two cell lines include upregulation of proapoptotic proteins such as Bax and depletion of Bcl2 expression, which was antiapoptotic protein, leading to an enhancement in Bax-to-Bcl2 ratio. Increased Bax-to-Bcl2 ratio resulting in the distribution of cytochrome C, and activation of caspase cascade and the internal pathway of apoptosis. Overall, based on these findings, the total extract of N. sativa is more effective in causing morphological changes and apoptosis and reducing viability in RCC than its fractions. Accordingly, it is offered that N. sativa total extract has more significant effects in the ACHN cell line. Consistent with our results, another study displayed that the total extract of N. sativa causes more marked proliferation inhibition in colon, oral, and prostate cancer cells in comparison with its fractions and pure active ingredient. In traditional and folklore medicine, it is also believed that whole plant extract, which is composed of several components, is more effective and less toxic compared with the active ingredients, which are isolated from the herbal extract. According to these findings, it is also confirmed that the effects of medicinal plants are the result of materialistic existence and the totality of their structure and are not just related to one particular chemical component. Moreover, it is suggested that components of N. sativa total extract besides having positive synergistic effects for greater efficacy have elements that neutralize the side effects. Thus, it is postulated that the fractionation of the plant total extract disrupts the plant's integrity and could reduce its therapeutic effect or increase its side effects.
| Conclusion|| |
Our findings demonstrated that N-butanol and dichloromethane fractions of N. sativa have more marked inhibitory effects with respect to morphological changes and cell viability, in the ACHN cell line than in the GP-293 cell line. Moreover, the effects of total extract were more pronounced than those of the two fractions, suggesting that anticancer agents are not concentrated in these fractions and that total N. sativa extract is a more potent anticancer agent compared with its fractions. However, the exact mechanisms involved in these actions remain to be elucidated.
Financial support and sponsorship
This study was part of an MSc thesis that was financially supported by Mashhad University of Medical Sciences (project code: MUMS 900198).
Conflicts of interest
There are no conflicts of interest.
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