Verso cDNA synthesis kit (AB1453A, Thermo Scientific), TRIzol Reagent (T9424, Sigma Aldrich), Taq Polymerase (MBT060A, Himedia), ready Mix dNTP (MBT078, Himedia), caspase-3 antibody (#9661, Cell signaling), BCL-2 antibody (SC-7382, Santa Cruz Technology), actin antibody (A02066, Sigma Aldrich), WesternSure-Premium Chemiluminescent substrate (WesternSure-Li-COR-Part No: 926-95000).

The human breast cancer MDA-MB-231 (metastatic) and MCF-7 (non-metastatic) cell lines, and cervical cancer SiHa cell line were procured from NCCS cell repository, Pune, India. J774A cell (Macrophage cell line) was obtained from Dr. Vijay Kumar Prajapati, Department of Biochemistry, Central University of Rajasthan, India. All cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (RM1112, Himedia) and maintained at 37°C in a humidified incubator with 5% CO₂.

Mononuclear cells were isolated from human peripheral blood by using a simple and rapid density gradient centrifugation technique using Ficoll-Paque (Sigma-F5414-50ML) methodology established by Boyum (1968) and Boyum (1976). The isolation was done according to the manufacturer’s protocol. Cells (0.5 × 10⁵ cells) were seeded in 96 well culture plate in DMEM supplemented with 10% FBS and incubated for overnight at 5% CO_2, and treated with increasing concentration (mentioned in other cell lines) by ethanolic and acetone plant extracts respectively for 24 h. Blood samples from two healthy volunteers were taken and mixed before isolation of PBMC. Written consent was obtained from the participants, and they were informed about the use of blood in this study. Moreover, the work related to blood samples had been conducted by following the regulation of Institutional Ethical Committee at Central University of Rajasthan and, this study was approved by Institutional Ethical Committee.

The plant Citrullus colocynth was obtained from a rural area of India [Jaisalmer (26.9157° N, 70.9083° E), Rajasthan, India]. The taxonomic name of this plant had been confirmed by Dr. Amit Kotia, Department of Botany, University of Rajasthan, India. The pulp was isolated from fruits, dried and crushed, using a mixer grinder. One gram of dried pulp powder was suspended in 10 ml of ethanol and acetone, followed by solvent extraction at 50°C for 72 h. The solvent fractions were evaporated using a rotary-evaporator at 42°C to get the crude products. The crude extracts obtained were then dissolved in ethanol and acetone and kept at -20°C for further use.

Cell viability was measured by MTT assay (MB186, Himedia) described earlier (Ghosh-Choudhury et al., 2010; Mehta et al., 2015). In brief, cancer cells (5000 cells/well) were seeded in 96 well tissue culture plate in DMEM supplemented with 10% FBS at an atmosphere of 5% CO₂ at 37°C. After 24 h of seeding, cells were separately incubated with different concentrations [25, 100, and 250 μg/ml; equivalent to 1, 4, and 10 μl of solvents (ethanol/acetone)] of fruit pulp ethanol extract (PEE) and pulp acetone extract (PAE) for 24 h. Control cells were similarly incubated with equal volume of the solvents as in the experimental wells. At the end of the incubation period, 10 μl of MTT solution (5 mg/ml) was added to each well and the plate was incubated at 37°C for 1 h. The formazan crystals formed were solubilized by adding DMSO, and subsequently, absorbance was measured at a wavelength of 530 nm (Studzinski, 1995; Moongkarndi et al., 2004; Ghosh-Choudhury et al., 2010; Mehta et al., 2015). The effective absorbance was calculated for the experimental wells with respect to corresponding control wells. LC₅₀ (Lethal concentration at which 50% cells are killed) at 24 h duration was calculated using MTT data set for different cancer cell lines (Zhang et al., 2007).

Cell migration was determined by scratch assay as described earlier (Mandal et al., 2010b, 2011b). In brief, cells (2 × 10⁵ cells/plate) were seeded in 35 mm tissue culture plates and allowed to grow till they reach 95% confluency. A cell scratch-wound was generated using a micropipette tip, and washed once with PBS buffer to remove the floating cells. Cells were then separately treated with PEE and PAE (25 μg/ml) for 24 h in experimental plates, and equal volume of respective solvents was added in control plates. Cells were visualized under an inverted microscope (Carl-Zeiss), and cell migration was assessed by measuring gap sizes in multiple fields and the relative area calculated using ImageJ, and statistically analyzed (Hirsch et al., 2009).

Transcript levels of several genes were measured by semi-quantitative RT-PCR analysis as described previously (Mandal et al., 2010a; Chowdhury et al., 2017). In brief, breast cancer cells (2 × 10⁵ cells/plate) were plated in 35 mm tissue culture plates in DMEM supplemented with 10% FBS at an atmosphere of 5% CO₂ at 37°C. After 90-100% cell confluency is reached, the cells were treated with different test factors (such as PEE and PAE (50 μg/ml)) for 24 h. Total RNA was extracted using TRIzol reagent as described earlier (Mandal et al., 2010a, 2011a, 2016a; Ghosh-Choudhury et al., 2013). First stand cDNA synthesis was performed using 1 μg of total RNAs using the Verso cDNA synthesis kit, according to manufacturer’s instruction. Semi-quantitative PCR was performed using this cDNA as template and gene specific primers in a ProFlex PCR system (Applied Biosystem by Life Technologies). The list of primers used in this study is given in Supplementary Table S1. Densitometric analysis of the PCR bands was done using ScnImage (Chowdhury et al., 2017).

Methodology of western blotting has been described previously (Chowdhury et al., 2017). In brief, cells were seeded at a density of 2 × 10⁵ cells/plate in 35 mm tissue culture plates. Cells were treated with different test factors and harvested after 24 h of treatment. Protein extraction was carried out in RIPA buffer and subsequently, cell lysates were centrifuged at 12,000 × g at 4°C for 20 min. The protein concentration was measured by Bradford reagent (BIORAD), according to manufacturer’s instruction. Equal amounts of whole cell lysates were resolved by 12% SDS-PAGE, and transferred to PVDF membrane. This PVDF membrane was incubated with primary antibody (Anti-caspase-3 antibody (#9661, Cell signaling), Anti-BCL-2 antibody (SC-7382, Santa Cruz Technology), Anti-actin antibody (A02066, Sigma Aldrich) used at 1:1000 dilution) overnight at 4°C. After washing, the PVDF membrane was further incubated with HRP-conjugated secondary antibody (used at 1:10000 dilution) for 1 h at room temperature. The washed membrane was further incubated with chemiluminescent substrate and subsequently, the blot was scanned by C-DIGIT Blot Scanner (Li-COR-Model: 3600).

Cells were seeded at a density of 500 cells/well in a 12 well tissue culture plate in DMEM with 10% FBS at 37°C incubator with 5% CO₂ for 24 h. Cells were then treated with PEE and PAE extracts (25 μg/ml), and incubated under same conditions for 5 days. Next, colonies were fixed with methanol and were stained with crystal violet solution (Hirsch et al., 2009). Photographs of stained plate were taken by a camera. Photographs of colonies were also taken by a bright field inverted microscope (Carl-Zeiss).

For performing soft agar assay (Sebolt-Leopold et al., 1999), MDA-MB-231 cells (10,000 cells/plate) were mixed with 2X DMEM containing 0.6% (w/v) agar and layered with 2X DMEM containing 1.2% base agar medium in 35 mm culture plates. 25 μg/ml of PEE and PAE was added in 300 μl of DMEM to the top agar and cells were allowed to grow for 8 days. The top 300 μl medium was replaced with fresh medium with or without test factors at intervals of every 3 days. The morphology and size of the spheres were visualized using bright field inverted microscope (Carl-Zeiss) and photographed. Subsequently, spheres were manually counted under the microscope.

Solvent Extraction of Citrullus colocynthis fruit pulp was done by ethanol and acetone. Dried extracts were dissolved in methanol followed by GC-MS/MS analysis using Thermo GC 1300 and “TSQ 8000” Triple quadrupole GC-MS/MS SYSTEM with auto sampler Al 1310. The Gas Chromatography 1300 was used with a fused GC column TG-5MS AMINE. The column length was of 30 m with an internal diameter coated film of 0.25 μm, with the flow rate of 10 ml/min and the condition kept were as follows: PTV Temp. Program: 70°C, hold 2.00 min, 10°C/min to 270°C, hold 8 min. Carrier gas helium flow rate was 1 ml/min and split ratio of 1:50. GC was equipped with an auto sampler AI 1300 and sample volume loaded was 1 μl. Eluates were automatically passed into the mass spectrometer. Mass Spectrum analysis was conducted using TSQ-8000 with a transfer line temperature of 280°C and ion source temperature of 230°C in EI mode. Mass scan time was 4 min with feature of full Scan MS. The mass spectrum was also equipped with a computer fed NIST mass Spectra data library.

Chemical constituents of the extracts were identified by matching the peaks with Computer NIST MS libraries and confirmed by comparing mass spectra of the peaks and those from literature. List of 50 compounds isolated from the extracts are listed in Supplementary Tables S2, S3. Scifinder was used to analyze the compounds and thus interpret the literature available for them.

Statistical analysis was done by GraphPad Prism (Ghosh-Choudhury et al., 2010; Mandal et al., 2011a,b, 2016b). A p-value of < 0.05 was considered to be statistically significant. All values were represented as mean ± standard error mean of three measurements.

To test the anticancer potential of colocynth fruit pulp extracts (prepared separately by solvent ethanol and acetone), cell viability was measured by MTT assay. For this, different cancer cells, including breast cancer cell lines, MCF-7 and MDA-MB-231, and cervical cancer cell line, SiHa, were separately treated with increasing concentrations (range 25–250 μg/ml) of PEE and PAE for 24 h (Figures 1A–C, 2A–C). MTT assay result showed noteworthy reduction in the number of viable cells in a dose dependent manner for both PEE and PAE treatment when compared to their respective solvent controls. To determine the effectiveness of the pulp extracts in these cancer cells, LC₅₀ values (using MTT data) were also calculated. In case of ethanol extract, LC₅₀ (μg/ml) values were 142 ± 16, 105 ± 8.3 and 157 ± 4.3 for MDA-MB-231, MCF-7 and SiHa cell lines respectively (Table 1). For acetone extract, these values were 140 ± 0.8, 94 ± 6.4 and 121 ± 4.6 respectively. These findings indicated relatively higher efficacy of PAE in cancer cell lines compared to PEE. Moreover, MCF-7 cells showed higher sensitivity to both PAE and PEE as compared to MDA-MB-231 cells. LC₅₀ values of both the extracts in PBMC and J774A cell line were >300 μg/ml (Table 1). Furthermore, microscopic visualization indicated a cytotoxic effect of the extracts upon these cancer cell lines (Figures 1D–F, 2D–F). All these findings suggested that the fruit pulp extracts induced reduced viability of cells that is presumably caused by inhibiting cell proliferation and inducing cytotoxicity.

To examine the influence of these pulp extracts on cell apoptosis, RT-PCR analysis was performed to compare the transcript levels of anti-apoptotic BCL2 and BCLXL and apoptotic BAX genes, using total RNA of MDA-MB-231 cells with or without treatment with ethanol and acetone extracts (50 μg/ml). Both PEE and PAE considerably impeded BCL2 and BCLXL expression with simultaneous upsurge in BAX expression (Figures 3A–D and Supplementary Figures S1A–C,E–G). Moreover, expression of apoptotic caspase 3 gene was augmented upon treatment with both PAE and PEE (Figures 3E,F and Supplementary Figures S1D,H). Furthermore, western blot results demonstrated increased level of cleaved caspase 3 (Figures 3G,H) with a concomitant decrease in the protein level of anti-apoptotic gene BCL2 (Figures 3I,J) for both ethanol and acetone extract treated cells compared to control. These observations led us to propose an apoptotic activity of fruit pulp extracts in breast cancer cells.

A common problem faced by a large percentage of cancer patients is development of metastasis if cancers are not detected in early and/or not treated with a suitable therapy. Migration of cancer cells is a critical step in the metastatic cascade (Chaffer and Weinberg, 2011). Thus, we investigated the effect of pulp ethanol and acetone extracts on the migratory property of cancer cells by performing scratch assay. Scratches were made on cell monolayer by a small pipet tip, and subsequently, cells were incubated with or without pulp extracts. Microscopic photos indicated migration of cells to close the induced scratch after 24 h in control MDA-MB-231, MCF-7 and SiHa cells (Figures 4A–C, 5A–C). Cells treated with both ethanol and acetone extracts (25 μg/ml) showed substantial prevention of cell migration as evident from the unfilled gap areas between the two ends of the scratches that were larger in cases of pulp extracts as compared to control. The data displayed in Figures 4D–F, 5D–F denoted a significant inhibition of cell migration in response to treatment with both ethanol and acetone extracts.

Transition from epithelial to mesenchymal phenotype is a critical step for metastasis (Chaffer and Weinberg, 2011). To examine the effect of fruit pulp extracts on prevention of EMT of metastatic MDA-MB-231 cells, we analyzed mRNA levels of several EMT markers by RT-PCR. Interestingly, we observed a decline in expression of mesenchymal markers, Vimentin and N-cadherin, with a concurrent increase in the expression of epithelial marker keratin-19 in treated samples (Figures 6A,B and Supplementary Figures S2A–C,F–H). Moreover, both PEE and PAE treatment strongly inhibited expression of the mesenchymal phenotype inducing transcription factors, Zeb1 and Zeb2 (Figures 6A,B and Supplementary Figures S2D,E,I,J). These observations clearly indicate a restraining impact of these pulp extracts on EMT of breast cancer cells.

Accumulating evidences suggest that cancers develop from cancer initiating cells or cancer stem cells (CSCs) (Reya et al., 2001; Singh and Settleman, 2010). Although CSCs comprise of a small percentage of the tumor population, their resistance to contemporary chemo- and radiation- therapy is a leading cause for tumor recurrence and metastasis (Li et al., 2007; Singh and Settleman, 2010). Thus, we investigated the effect of colocynth pulp extracts on the stemness property of metastatic MDA-MB-231 cells by colony formation assay. In agreement with previous observations, the number of colony formed was markedly reduced when cells were treated with pulp extracts (25 μg/ml) as compared to control, indicating an inhibitory role of both PEE and PAE on cancer stemness (Figures 7A–D). To further support these results, we carried out soft agar sphere formation assay. Microscopic visualization documented formation of a great number of spheres with large size after 8 days of cell culture in the control plates. The number as well as the size of such spheres was, however, greatly diminished when cells were incubated with both PAE and PEE (25 μg/ml) as compared to control (Figures 8A–D). These results show that fruit pulp extracts might attenuate stemness property of cancer cells. To verify these findings, we further examined the effect of these pulp extracts on the expression of stemness associated genes CD44 and BMI-1. RT-PCR analysis documented that transcript levels of both CD44 and BMI-1 genes (Al-Hajj et al., 2003; Liu et al., 2006) were significantly lowered in MDA-MB-231 cells when treated with both PAE and PEE as compared to control (Figures 8E,F and Supplementary Figures S3A–D). Together, these data clearly suggested that fruit pulp extracts (PEE and PAE) obliterate the stemness property of breast cancer cells.