D-carvone induced ROS mediated apoptotic cell death in human leukemic cell lines (Molt-4)

The immature lymphoid cells with chromosomal structural and numerical abnormalities cause the acute lymphoblastic leukemia (ALL). This hematologic disorder constitutes about 25% of cancer prognosis among children and adolescents. D-Carvone, a monocyclic monoterpene obtained from the essential oils extracted from plants is reported to possess the various biological activities. The present study was aimed to investigate the anticancer potential of D-Carvone against the human leukemic Molt-4 cells. The cytotoxicity of DCarvone was analyzed by MTT assay. The level of lipid peroxidation and antioxidants were determined. The intracellular ROS, MMP and apoptosis were demonstrated by fluorescent staining techniques. The MTT assay revealed that the D-Carvone treatment suppressed the viability of Molt-4 cells and the IC50 was determined at 20 µM/ml. The D-Carvone treatment was increased the oxidative stress and reduced the level of antioxidants in the Molt-4 cell lines. The increased intracellular ROS, apoptotic cell death, and diminished MMP was noted in the D-Carvone treatment. In the Molt-4 cells, D-carvone induced the apoptosis in a time and dose dependent manner by the activation of caspases-8, -9 and -3. Thus, data provide insights for the clinical application of D-Carvone in the treatment of blood cancer Molt-4 cells. Our study suggests the therapeutic potential D-Carvone for the treatment of leukemia in future.


Background:
The resources for treatment of cancer are minimal in developing nations [1]. Acute lymphoblastic leukemia (ALL) is a heterogeneous hematological malignancy caused by lymphoid cell proliferation and abnormal differentiation in bone marrow, peripheral blood and various other organs [2]. The high degree of heterogeneity is attributed to diverse genetic alterations that were acquired over a period of time. These alterations include both numerical and structural rearrangement of chromosomes, gain and loss of genomic regions and single nucleotide alterations. Approximately 75 to 80 % children develop ALL compared to adults. Antileukemic drugs and improved multimodality treatment has increased the 5 year survival rate of children with ALL above 85 % in developed countries [3] but majority take a turn for the worse within 2 years of diagnosis resulting in a five year survival rate is reduced to 10 % [4]. In the view of the above facts investigation of therapeutic substances that are effective in reducing the carcinogenicity of mutagens gained importance [5][6][7]. Antioxidants are important for their protective role followed by the free radicals induced damages, which may lead to several cancers including blood cancer [8]. Most of the chemotherapeutic agents currently employed are directly or indirectly derived from the natural sources. Among these natural sources secondary metabolites from plants and their derivatives have been proved their potentials as effective anticancer drugs [9,10]. Monoterpenes present in plant essential oils contribute to the aroma of plants [11,12]. D-carvone is one such monoterpenic ketones that is present in various essential oils extracted from plants and has been utilized in food and pharmaceutical industry [13,14]. Literature also reports that this bioactive compound possesses antimicrobial, antioxidant, antitumor and anticonvulsant properties [15][16][17]. D-carvone also possessed the neuroprotective [18], anticancer [19,20], antiarthritis [21], and anti-ulcerative colitis [22] activities. Therefore, it is of interest to document data on the D-carvone ROS mediated apoptotic cell death in human leukemic cell lines (Molt-4).

Cell culture, proliferation and treatments:
Human ALL (Molt-4) cells were purchased from the ATCC, USA. Cells were developed in DMEM medium supplemented with heat inactivated FBS (10%) and antibiotics and maintained in the CO2 incubator at 37 o C in an atmosphere of air (95%) and CO2 (5%) with 98% humidity.

MTT cytotoxicity assay:
The cytotoxicity effects of D-carvone against the Molt-4 cells were analyzed by the method of Mosmann et al. (1983) [23]. Briefly, 6 × 10 3 cells per well were seeded in 96 well plate and were treated with different concentrations of D-carvone (5 µM to 30 µM) for 24 h. 20 µl of MTT dye (2.5 mg/ml) was added and incubated for 3 h before the termination of the experiment. After that, the culture plate was incubated for 4 h at 37°C and formulated formazan crystals were dissolved by the addition of 150 µl of DMSO. Finally, the absorbance was measured at 570 nm with reference wavelength at 620 nm. The percentage of cell viability was calculated at 50% inhibitory concentration (IC50) was determined.

Measurement of intracellular ROS:
The intracellular accumulation of ROS was monitored using DCFH-DA staining technique. Deacetylation within the cell leads to the binding of dye with intracellular radicals, generated in a quantitative manner and it is converted into its fluorescent product DCF. Molt-4 cells treated with D-carvone for 24 h were harvested ©Biomedical Informatics (2021) and re-suspended in PBS (pH 7.4). DCFH-DA solution (10 µM) was added to 2 x 10 5 ml of cell suspension. The mixture was incubated at 37ºC for 30 min. Cells were then washed twice and re-suspended in PBS. The fluorescence intensity was measured spectroflurometrically with excitation and wavelengths ranging from 485 nm and 530 nm respectively.

Measurement of MMP:
Molt-4 cells were seeded in 6-wellplate and incubated with different concentrations of D-carvone (15 and 20 µM/ml) for 24 h. Rh-123 is a fluorescent probe used to estimate the depolarization of MMP. Rh-123 dye was added and incubated for 30 min at 37ºC. After incubation the cells were washed with PBS and observed under fluorescence microscope (Labomed, USA). Fluorescence intensity of the captured images was analyzed by Image J software. Fluorescence intensity of the captured images was taken using a blue filter (450-490 nm).

Observation of morphological and nuclear changes AO/EB staining
Briefly, the AO/EB (AO: 100Ĵg/ml, EB: 100Ĵg/ml) stain solution was added to D-carvone (15 and 20 µM/ml) treated Molt-4 cells placed in a cover slip. Placing the cover slip over it spread the dye. The stained slides were then incubated at room temperature for 5 min. The apoptotic cells were visualized for green fluorescence, which was counted using an upright fluorescent microscope at 40x magnification.

PI staining:
After treatment with the (15 and 20 µM/ml) of D-Carvone for 24h, the traces of medium and serum were removed from Molt-4 cells and cleansed with PBS. The cells were permeabilized using 50µl acetone and methanol in 1:1 ratio at -20°C for 10 min. Then 10µl of PI was added and spread by placing the cover slip over it and incubated at 37°C for 30 min in dark. Finally the PI stained cells were observed under the fluorescence microscope.

Statistical Analysis:
Data were illustrated as mean ± SD of triplicate measurement. Statistical evaluations were assessed using the SPSS software. Significance level was calculated by using one-way ANOVA followed by DMRT test. Results are considered as statistically significant if p < 0.05.

Effect of D-carvone induced apoptotic cell death in Molt-4 cells:
The apoptotic cell death in the D-Carvone (15 µM and 20 µM) treated Molt-4 cells was investigated by dual (AO/EtBr) staining technique. Figure 5A revealed that the untreated control cells displayed AO stained green fluorescence; interestingly, the D-Carvone treated Molt-4 cells were revealed the intense EtBr stained orange fluorescence that demonstrating the apoptotic cells. Hence, it was clear that the D-Carvone has the potential to stimulate apoptosis in blood cancer cells. Both concentrations (15 and 20µg) of the D-Carvone has enthused the apoptosis in the human blood cancer Molt-4 ( Figure 5).

Effect of D-carvone induced apoptosis in Molt-4 cells:
The PI was executed to differentiate the necrotic cells from viable cells.    ©Biomedical Informatics (2021)

Discussion:
Nutrients have a very important role in maintaining normal health. Dietary antioxidants are potential adjuvant in cancer therapy since they are capable of inducing apoptosis in the cancer cells [28]. Apoptosis or programmed cell death is an essential mechanism for the development and homeostasis of multicellular organism for eliminating unwanted cells [29]. Failure or inefficient apoptosis is an important factor of tumorigenicity and induction of apoptosis is the target for cancer therapy [30]. The major phytochemicals such as flavonoids, terpenoids, carotenoids and selenium were reported for their anticancer property against numerous cancers [32]. Previous literatures demonstrated the anti-carcinogenic properties of several monoterpenes in experimental models such as liver, melanoma, breast and prostate cancer [31]. Carvone a monoterpene ketone is found predominantly in essential oils of spearmint and caraway is used as an odorant and flavor accompaniment and forms a common ingredient in human diet Our findings from this study coincide with the above findings. MMP was investigated to understand the mechanism of apoptosis in D-carvone treated Molt-4 cell lines. The control cells were found with intense green fluorescence, which indicated that there were no changes in mitochondrial transmembrane potential. But the cells treated with D-carvone showed loss of green fluorescence due to depolarization of mitochondrial membrane. The effect of D-carvone on the MMP of HT-29 and SW480 colon cancer cell lines with similar observation to the present study was reported [48]. In the non-apoptotic cells the dye were accumulated within the mitochondria and thus exhibiting a bright green color. Decreased accumulation of the dye in the mitochondrial indicates the collapse in the MMP. This may be correlated with high ROS generation. ROS triggers the apoptotic signaling by inducing depolarization of mitochondrial membrane, which results in, increased LPO byproducts (TBARS) and decreased activity of antioxidant enzymes (SOD and CAT). These effects are attributes to D-carvone induced ROS generation. The result from the present study was in concordance with earlier studies on colon cancer cells and Hela cells [49]. In order to elucidate the levels of antioxidant enzyme status on the Molt-4 cell lines, the activities of SOD, CAT, GSH and MDA contents were measured. The possible antitumor and antioxidant activity of D-carvone in Molt-4 cell lines were evaluated by measuring the endogenous antioxidant levels. The high accumulation of reactive oxygen species during the process of carcinogenesis may play an important role in causing oxidative damage. Hence there might be an increase or decrease in the antioxidant enzymes. The present study revealed that the activities of SOD and CAT was higher in the untreated MOLT-4 cells (control) compared to D-carvone treated Molt-4 cell lines. A similar observation of higher activity of SOD and CAT were reported in hepatoma (HepG-2) cell lines [44]. The increase in activity of SOD and CAT was reported to be 2 fold and 4.3 fold respectively [50,51]. These findings were in agreement with our present results that SOD and CAT had increased activity in Molt-4 cells compared to Dcarvone treated cell lines. GSH content was found decreased and TBARS content was found increased in D-carvone treated MOLT-4 cell lines. Low levels of GSH were observed in chronic alcoholic liver disease and liver cancer. Such observation of low GSH could be due to alterations in defense system in tumor cells [50]. The study revealed that the anti-tumor activity of D-carvone might influence the status of antioxidant enzymes in Molt-4 cells. The Higher activities of SOD and CAT was reduced significantly with the treatment of D-carvone, Thus D-carvone might have played the ©Biomedical Informatics (2021) role of antioxidants such as SOD and CAT in eliminating the superoxide radicals and accumulation of H2O2 in Molt-4 cell lines. The activation of caspase protein family begins with caspase-3 that initiates apoptosis by activating caspase-8 and -9 [52,53]. Down regulation of caspase 3 and 9 was reported in DMBA induced skin cancer. D-carvone 20 mg/kg of bw, was recommended as optimal dose for DMBA treated skin cancer in mice [17]. Furthermore, the effect of D-carvone pre-treatment on the expression of the apoptosis-related proteins was determined in our data. As shown in Figure 7, D-carvone pre-treatment abrogated cytochrome c release, as well as the activation of caspase-3, -8 and -9. In agreement with the preceding results of AO/EB and PI staining, these findings indicated that the cytotoxic effect of D-carvone in Molt-4 cells is mediated through apoptotic induction, as well as mitochondrial dysfunction involved in ROS production.

Conclusion:
We document data on the D-carvone induced ROS mediated apoptotic cell death in human leukemic cell lines (Molt-4) for further consideration.
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