Potent anticancer activity of (Z)-3-hexenyl-β-D-glucopyranoside in pancreatic cancer cells

This current study reports, for the first time, on the potent cytotoxicity of (Z)-3-hexenyl-β-D-glucopyranoside, as well as its cellular and molecular apoptotic mechanisms against Panc1 cancer cells. The cytotoxicity of three compounds, namely (Z)-3-hexenyl-β-D-glucopyranoside (1), gallic acid (2), and pyrogallol (3), which were isolated from C. rotang leaf, was investigated against certain cancer and normal cells using the MTT assay. The cellular apoptotic activity and Panc1 cell cycle impact of compound (1) were examined through flow cytometry analysis and Annexin V-FITC cellular apoptotic assays. Additionally, RT-PCR was employed to evaluate the effect of compound (1) on the Panc1 apoptotic genes Casp3 and Bax, as well as the antiapoptotic gene Bcl-2. (Z)-3-hexenyl-β-D-glucopyranoside demonstrated the highest cytotoxic activity against Panc1 cancer cells, with an IC50 value of 7.6 µM. In comparison, gallic acid exhibited an IC50 value of 21.8 µM, and pyrogallol showed an IC50 value of 198.2 µM. However, (Z)-3-hexenyl-β-D-glucopyranoside displayed minimal or no significant cytotoxic activity against HepG2 and MCF7 cancer cells as well as WI-38 normal cells, with IC50 values of 45.8 µM, 108.7 µM, and 194. µM, respectively. (Z)-3-hexenyl-β-D-glucopyranoside (10 µM) was demonstrated to induce cellular apoptosis and cell growth arrest at the S phase of the cell cycle in Panc1 cells. These findings were supported by RT-PCR analysis, which revealed the upregulation of apoptotic genes (Casp3 and Bax) and the downregulation of the antiapoptotic gene Bcl-2. This study emphasizes the significant cellular potency of (Z)-3-hexenyl-β-D-glucopyranoside in specifically inducing cytotoxicity in Panc1 cells. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00210-023-02755-4.


Introduction
Pancreatic cancer is one of the most fatal malignant tumors, posing a significant challenge in early diagnosis due to the absence of clear signs and symptoms.In advanced stages, pancreatic cancer exhibits a high mortality rate, with approximately 97% of patients expected to succumb within 5 years of diagnosis (Mario et al. 2018;Zhang et al. 2018).Disturbingly, the incidence, prevalence, and mortality rates of pancreatic cancer have all witnessed a global surge over the last 25 years, and projections indicate a further alarming increase of approximately 1.97-fold by 2060 (Mario et al. 2018).
Anticancer drugs continue to be a vital element in the treatment protocol for pancreatic cancer, whether used as standalone therapies or in conjunction with other modalities such as surgery, radiotherapy, and immunotherapy (Pliarchopoulou and Pectasides 2009;Neoptolemos et al. 2018).
Moreover, a variety of plant metabolites have exhibited potent cytotoxic activity against Panc1 cancer cells through diverse mechanisms.For example, berberine, a natural anticancer agent, has been identified as an inhibitor of Panc1 proliferation and an inducer of apoptosis (Rauf et al. 2021).Damnacanthal, isolated from Garcinia huillensis, has demonstrated the ability to induce necrotic death in Panc1 cells (Dibwe et al. 2012).Furthermore, curcumin, rhein, ellagic acid, embelin, metformin, and eruberin A are additional plant metabolites derived from various plant sources that exhibit cytotoxic effects against Panc1, with their respective mechanisms of action confirmed (Ramakrishnan et al. 2020).These findings suggest that further exploration of plant extracts and metabolites may lead to the discovery of novel sources of anticancer agents against Panc1, making them an area of significant scientific interest.
Further research is warranted to delve into the potential biological activities and therapeutic applications of this natural metabolite.

Plant materials
The leaves of Calamus rotang L. (Fig. 1) were gathered from the Aswan Botanical Garden, located in Aswan, Egypt, in October 2018.Dr. Amr M. M. Mahmoud, the Director of Aswan Botanical Garden at the Horticultural Research Institute, Agriculture Center, Egypt, completed the taxonomical identification of the plant.A voucher specimen with the number A20220906 was deposited at the herbarium of the Department of Pharmacognosy, School of Pharmacy, Assiut University, Egypt.

Cell culture
The cell lines PANC-1 (pancreatic carcinoma), MCF7 (breast carcinoma), HepG2 (hepatocellular carcinoma), and WI-38 (normal lung fibroblast cells) were obtained from the American Type Culture Collection (ATCC) located in Manassas, Virginia, United States.These cell lines were cultured in DMEM (Invitrogen, Life Technologies, Rockville, MD, USA) medium supplemented with 10% FBS (Fetal Bovine Serum, Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin (Sigma-Aldrich, Louis, MO, USA).The cells were maintained at 37°C in a 5% CO 2 and 95% humidity environment for a maximum period of two weeks to ensure their viability for further experimentation.Cell detachment was achieved using a solution containing 0.25% (w/v) trypsin (Sigma-Aldrich, Louis, MO, USA) and 0.53 mM EDTA ((Sigma-Aldrich, Louis, MO, USA), followed by re-cultivation in fresh media.

MTT assay
The cultivated cells were seeded in 96-well plates at a concentration of 1 × 10 4 cells/100 μL per well (Naik et al. 2014).The cultured plates were then incubated for 24 hours at 37°C.Stock solutions of the isolated compounds (1-3) and staurosporine (reference compound) were prepared at a concentration of 1 mg/mL in 10% DMSO (Sigma-Aldrich, Louis, MO, USA) in ddH 2 O.The tested concentrations of the compounds (1, 10, 30, and 100 µg/mL) in 0.01 -0.1% DMSO in ddH 2 O were prepared by diluting the stock solutions with double-distilled water (ddH 2 O).After a 24-hour incubation of cells in 96-well plates, the media were replaced, and the tested concentrations of each compound, along with staurosporine, were added in triplicates (Esharkawy et al. 2022).A negative control using 0.1% DMSO in ddH 2 O was also included.The treated cells were further incubated for 48 hours at 37 °C.The media of the treated and control plates were removed and replaced by fresh media containing MTT (Sigma, Louis, MO, USA) reagent in a concentration of 1 mg/mL (Ponnusamy et al. 2016), then incubated for 2 hours.The viability of the cancer cells was determined by measuring the amount of formazan formed by viable cells.The produced formazan was solubilized by adding 100 µL 10% DMSO in ddH 2 O per each treated and control well.The plates were gently shacked for 5 min.The intensity of the produced colour was measured using an ELISA plate reader (Bio-Tek EL 800, Agilent technology, Santa Clara, CA, USA) at wavelength of 570 nm.The percent of cells viability was calculated by the following equation:

AA Average of triplicate absorbances for each sample concentration
Calibration curves of the tested and reference compounds were then prepared to calculate the IC 50 (the concentration that inhibits 50% of cancer cells) (Esharkawy et al. 2022).

Flow cytometry analysis of the cell DNA contents in Panc1 cells treated with (Z)-3-hexenyl-β-D -glucopyranoside
All reagents and kits for this assay were obtained from Abcam (Abcam Technology, Boston, MA, USA).The detection of DNA cell contents and cell cycle status was % viability =AA 570 of treated − AA 570 of blank ∕AA 570 of control − AA 570 of blank × 100 conducted following the established method (Xu et al. 2001).To provide a brief overview, Panc1 pancreatic cancer cells (ATCC, Manassas, Virginia, USA) were cultured in a single-cell suspension in DMEM medium (Invitrogen, Life Technologies, Rockville, MD, USA).Subsequently, the cells were fixed in 66% ethanol (Sigma-Aldrich, Louis, MO, USA) and kept on ice for 2 hours.Afterward, the cancer cells were washed with a PBS (Sigma-Aldrich, Louis, MO, USA) solution (5 mL of 10X PBS + 45 mL water), re-cultured in fresh medium, and incubated at 37 ºC for 24 hours.A concentration of 10 µM of (Z)-3-hexenyl-β-D -glucopyranoside was added in triplicate, chosen based on previously reported data (Ponnusamy et al. 2016;Xu et al. 2001) for 24h.The second dose of 10 µM of (Z)-3-hexenyl-β-D -glucopyranoside was added to the Panc1 cells then incubated for 24 h.Following a 48-hour incubation period from the first dose, the cells were trypsinized, fixed, and stained with propidium iodide-RNase enzyme reagent (9.45 mL PBS + 500 μL 20X propidium iodide + 50 μL 200X RNase) from Abcam Technology, Boston, MA, USA.The intensity of propidium iodide fluorescence, and thereby the amount of cellular DNA in each stage of the Panc1 cell cycle, was quantified using a flow cytometer (Novocyte, Agilent technology, Santa Clara, CA, USA) with an excitation maximum of 493nm and an emission maximum of 636 nm.The incubation time for fluorescence quantification was 30 minutes.The experiment was repeated twice.

Annexin V-FITC cellular apoptotic assay
The reagents used in this assay included the Annexin V-FITC kit (Abcam Technology, Boston, MA, USA), 1X binding buffer (Abcam Technology, Boston, MA, USA), and propidium iodide (Bio Vision Research Products, Mountain View, CA, USA).Panc1 cells were seeded in 6-well plates and incubated at 37 ºC for 24 hours.Compound 1 ((Z)-3hexenyl-β-D -glucopyranoside) was then added to the cultivated Panc1 6-well plates at a concentration of 10 µM and incubated for 48 hours.Subsequently, the cancer cells were trypsinized and centrifuged for 10 minutes at 300 rpm.The resulting precipitate was resuspended in 500 µL of 1X binding buffer.Annexin V-FITC (5 µL) and propidium iodide (5 µL) reagents were added to the treated cancer cell plates and incubated for 5 minutes in the dark at room temperature.A flow cytometer (Novocyte, Agilent technology, Santa Clara, CA, USA) with an excitation maximum of 488 nm and an emission maximum of 530 nm was employed to measure the intensity of annexin-binding phosphatidylserine (PS) and consequently determine the amount of apoptotic cells (Koopman et al. 1994).

Statistical analysis
The mean standard error of three measurements was used to calculate the IC 50 of the tested reference compounds.ANOVA was used for statistical comparisons.P-values less than 0.05 were considered significant in the differences of treated cell lines by compounds 1-3 and reference stourosporine compared with the solvent control (0.1% DMSO).

Cytotoxic activity of compounds 1-3
The cytotoxic activities of the isolated compounds 1-3 are depicted in Figs. 3 and S2 and Table 1.In this study, we present the novel finding of the cytotoxic activity of (Z)-3hexenyl-β-D -glucopyranoside (1) (Figs.3A, B, and 4) against pancreatic (Panc1), hepatic (HepG2), and breast (MCF7) cancer cells, as well as WI-38 normal cells.It should be noted that compounds 2 and 3 have previously been reported as cytotoxic natural agents (Faried et al. 2007;Jiang et al. 2022;Maurya et al. 2010;Revathi et al.).In this study, we describe their cytotoxic activity against Panc1 cells, as shown in Table 2 and Fig. 3C.

Conclusion
The findings of this study strongly suggest that (Z)-3hexenyl-β-D -glucopyranoside (1) holds great potential as an effective cytotoxic compound against Panc1 cells.Its    These findings shed light on the promising cellular activity exhibited by (Z)-3-hexenyl-β-D -glucopyranoside (1) in inducing cytotoxicity, particularly in Panc1 cells.Consequently, it is essential to conduct further investigations to explore its potential in vivo anticancer activity against pancreatic carcinoma.Future studies should focus on evaluating the efficacy and safety of (Z)-3-hexenyl-β-