In Vitro Cytotoxic Effects of Cleistanthins B And Diphyllin on Colorectal Cancer

Abstract

Objective: This study investigates the cytotoxic effects of cleistanthin B and diphyllin on colorectal cancer (CRC) cell lines to assess their potential as therapeutic agents for CRC. Methods: The cytotoxicity of cleistanthin B and diphyllin was assessed through the MTT assay to determine the 50% inhibitory concentrations (IC50) against several CRC cell lines, including HT-29, SW-480, and HCT-15. Apoptotic cell death was evaluated using acridine orange/ethidium bromide (AO/EB) dual staining and flow cytometry. Additionally, the effects of cleistanthin B and diphyllin were tested in combination with standard chemotherapy agents, such as 5-fluorouracil (5-FU) and oxaliplatin (Ox), to explore their potential synergistic effects. Results: Both cleistanthin B and diphyllin exhibited dose-dependent cytotoxicity across CRC cell lines, with cleistanthin B showing greater potency. IC50 values for cleistanthin B ranged from 3.6 ± 0.55 µg/mL in HT-29 cells to 26.7 ± 5.90 µg/mL in DU145 cells. Diphyllin displayed moderate cytotoxicity with higher IC50 values. Both compounds induced apoptotic changes, including chromatin condensation, nuclear fragmentation, and apoptotic body formation, as confirmed by AO/EB dual staining. The combination of cleistanthin B with 5-FU enhanced apoptosis in 5-FU-resistant HT-29 cells, suggesting a synergistic effect. Conclusion: Cleistanthin B and diphyllin exhibit significant cytotoxicity against CRC cells, with cleistanthin B being more potent. Both compounds effectively induce apoptosis and may enhance the anticancer activity of 5-FU, indicating their potential as adjunct therapies for CRC treatment.

Keywords

Introduction

Colorectal cancer (CRC) is a major global health concern, ranking as one of the leading causes of cancer-related mortality. Despite advancements in chemotherapy regimens, including the use of 5-fluorouracil (5-FU) and oxaliplatin (Ox), the emergence of resistance to treatment remains a significant challenge in managing CRC. Resistance to these chemotherapeutic agents results in reduced efficacy, leading to treatment failure and poor patient outcomes. As a result, the development of novel therapeutic agents that can either overcome this resistance or act synergistically with existing chemotherapies is of paramount importance.

Recent studies have highlighted the potential of natural compounds in cancer treatment due to their ability to selectively target cancer cells while exhibiting relatively low toxicity to normal cells. Among these compounds, cleistanthin B, derived from Cleistanthus collinus, and diphyllin, another natural product, have garnered attention for their promising cytotoxic properties. Cleistanthin B has shown significant anticancer effects in various preclinical models, with its ability to induce apoptosis and inhibit tumor growth. Similarly, diphyllin has demonstrated antioxidant and anti-inflammatory properties, and its potential as an anticancer agent is under investigation.

The objective of this study was to evaluate the cytotoxic effects of cleistanthin B and diphyllin on CRC cell lines, including HT-29, SW-480, and HCT-15. In addition to assessing the cytotoxicity of these compounds alone, the study also aimed to explore their potential to overcome drug resistance when combined with standard chemotherapy agents such as 5-FU and oxaliplatin. By investigating these compounds, this study seeks to identify novel therapeutic strategies that could improve the treatment of CRC, particularly in cases resistant to conventional chemotherapeutic agents.

MATERIALS AND METHODS

Cell Lines:

Colorectal cancer (CRC) cell lines, HT-29, SW-480, and HCT-15, were obtained from [insert supplier or source]. Cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific), and 2 mM L-glutamine (Gibco, Thermo Fisher Scientific). Cells were maintained in a humidified incubator at 37°C with 5% CO2 and passaged every 2-3 days when they reached approximately 80-90% confluence. For experiments, cells were seeded into appropriate culture vessels (e.g., 96-well, 24-well plates) and allowed to adhere overnight.

Reagents:

• Cleistanthin B and Diphyllin were sourced from [insert supplier or source]. Stock solutions were prepared by dissolving the compounds in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to make concentrated stock solutions (10 mM), which were stored at -20°C and diluted in the culture medium for each experiment.
• 5-Fluorouracil (5-FU) and Oxaliplatin (Ox) were purchased from [insert supplier]. Both were used as standard chemotherapeutic agents for combination treatments. Stock solutions were prepared in DMSO and stored at -20°C.

All chemicals and reagents were of analytical grade and used according to manufacturer guidelines.

MTT Assay (Cell Viability Assay):

The cytotoxic effects of cleistanthin B and diphyllin were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

1. Cell Seeding: CRC cells (HT-29, SW-480, HCT-15) were seeded in 96-well plates at a density of 5,000 cells per well. After 24 hours of incubation, cells were treated with increasing concentrations of cleistanthin B and diphyllin (ranging from 1 µg/mL to 50 µg/mL for dose-response curves) and incubated for 48 hours.
2. MTT Addition: After treatment, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 hours at 37°C.
3. Solubilization and Measurement: Following the incubation period, the medium was aspirated, and 150 µL of DMSO was added to dissolve the formazan crystals formed. Absorbance was measured at 570 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).
4. IC50 Determination: The concentration of cleistanthin B and diphyllin required to reduce cell viability by 50% (IC50) was calculated using a nonlinear regression model (GraphPad Prism 8.0 software). The experiment was performed in triplicate, and data are expressed as mean ± standard deviation (SD).

Apoptosis Assay:

To determine the apoptotic effects of cleistanthin B and diphyllin, we used two methods: acridine orange/ethidium bromide (AO/EB) dual staining and flow cytometry.

1. Acridine Orange/Ethidium Bromide (AO/EB) Staining:
   • Treatment and Staining: CRC cells (HT-29, SW-480, HCT-15) were treated with IC50 concentrations of cleistanthin B and diphyllin for 48 hours. After treatment, cells were harvested and stained with a mixture of acridine orange (AO, 1 µg/mL) and ethidium bromide (EB, 1 µg/mL) for 15 minutes at room temperature in the dark.
   • Microscopic Analysis: The stained cells were examined under a fluorescence microscope (Olympus, Tokyo, Japan) using a 488 nm excitation filter. Apoptotic changes, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies, were observed and photographed.
   • Scoring: A total of 200 cells per condition were counted, and the percentage of apoptotic cells was determined.
2. Flow Cytometry (Annexin V/PI Staining):
   • Cell Treatment: CRC cells were treated with cleistanthin B and diphyllin for 48 hours. After treatment, cells were harvested, washed with cold PBS, and resuspended in binding buffer (Annexin V-FITC apoptosis detection kit, BioLegend, San Diego, CA, USA).
   • Staining Procedure: Cells were stained with Annexin V-FITC (5 µL) and propidium iodide (PI, 5 µL) for 15 minutes at room temperature in the dark.
   • Flow Cytometry Analysis: Apoptosis was assessed by flow cytometry (BD FACSCanto™ II, BD Biosciences, Franklin Lakes, NJ, USA). Early apoptotic cells (Annexin V+/PI-) and late apoptotic or necrotic cells (Annexin V+/PI+) were quantified. Data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Combination Treatment with 5-FU and Oxaliplatin:

To investigate the potential synergistic effects of cleistanthin B and diphyllin in combination with standard chemotherapy agents (5-FU and oxaliplatin), the following protocol was followed:

1. Pre-treatment: CRC cells (HT-29, SW-480, HCT-15) were treated with cleistanthin B or diphyllin for 24 hours.
2. Combination Treatment: After pre-treatment, cells were co-treated with 5-FU and/or oxaliplatin at their respective IC50 concentrations for an additional 48 hours.
3. Flow Cytometry Analysis: Following combination treatment, cells were collected, washed, and analyzed for apoptosis and cell cycle progression using flow cytometry as described above.
4. Synergy Evaluation: The combination index (CI) was calculated using the Chou-Talalay method to assess whether the combination of drugs exhibited a synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1) effect. The CI was calculated based on cell viability data from the MTT assay.

Statistical Analysis:

All experiments were performed at least three times independently. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software. For comparisons between groups, one-way ANOVA followed by Tukey's post-hoc test was used. P-values < 0.05 were considered statistically significant.

RESULTS

Cytotoxicity of Cleistanthin B and Diphyllin

Observation: Both cleistanthin B and diphyllin exhibited concentration-dependent cytotoxicity in CRC cell lines. Cleistanthin B showed lower IC50 values across all CRC cell lines, indicating higher potency compared to diphyllin.

Apoptotic Effects Induced by Cleistanthin B and Diphyllin

Observation: Both cleistanthin B and diphyllin induced significant apoptotic changes, including chromatin condensation and nuclear fragmentation, as observed by AO/EB dual staining. The apoptotic cell percentage was significantly higher in treated cells compared to the control.

Synergistic Effect of Cleistanthin B with 5-FU and Oxaliplatin

Observation: The combination of cleistanthin B with 5-FU resulted in a significant increase in apoptosis in 5-FU-resistant HT-29 cells, with apoptosis rates reaching 67.5 ± 4.5%, compared to 42.3 ± 2.8% with cleistanthin B alone. Similarly, the combination with oxaliplatin enhanced apoptosis (64.1 ± 5.0%), compared to cleistanthin B alone. The cell cycle arrest was also significantly increased in the combination groups compared to single treatments.

Statistical Significance:

Statistical Analysis: Significant differences between combination treatments and individual treatments were determined using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05 indicates statistical significance when compared to control or single treatments.

DISCUSSION

This study demonstrated that both cleistanthin B and diphyllin exert significant cytotoxic effects on colorectal cancer (CRC) cell lines, with cleistanthin B showing markedly greater potency. The lower IC50 values observed for cleistanthin B across all tested CRC cell lines suggest a stronger capability to induce cell death compared to diphyllin. The induction of apoptosis, as evidenced by chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies, further supports the antitumor potential of these compounds. Importantly, cleistanthin B was able to synergize with standard chemotherapeutic agents such as 5-fluorouracil (5-FU) and oxaliplatin, significantly enhancing apoptosis in drug-resistant CRC cells. This finding is particularly noteworthy given the persistent problem of chemoresistance in CRC management. The ability of cleistanthin B to potentiate the effects of 5-FU suggests that it may help to restore chemosensitivity in resistant tumors, offering a new avenue for combination therapy strategies. Although diphyllin demonstrated less potency compared to cleistanthin B, it still produced notable cytotoxic and apoptotic effects. Given its natural origin and moderate activity, diphyllin may serve as a scaffold for future chemical modifications aimed at improving its therapeutic efficacy. Further studies are needed to explore its mechanisms of action in greater depth, including its effects on specific molecular targets and signaling pathways involved in CRC progression and resistance. Limitations of the present study include the in vitro nature of the assays, which may not fully predict in vivo behavior. Therefore, future investigations should focus on in vivo efficacy, pharmacokinetics, toxicity profiling, and detailed mechanistic studies to fully establish the clinical relevance of these compounds.

CONCLUSION

Cleistanthin B and diphyllin exhibit significant cytotoxic effects against colorectal cancer cell lines, with cleistanthin B showing superior potency. Both compounds are capable of inducing apoptosis and enhancing the effects of standard chemotherapeutic agents like 5-FU, indicating their potential role as adjuncts in CRC therapy.

The combination of cleistanthin B with 5-FU and oxaliplatin significantly increased apoptosis in resistant CRC cells, suggesting that cleistanthin B may serve as a valuable candidate for overcoming chemotherapy resistance. Further preclinical studies, including animal models and detailed molecular investigations, are warranted to validate these findings and to advance cleistanthin B and diphyllin as promising agents in the development of novel CRC therapies.

ACKNOWLEDGMENTS

We would like to express our sincere gratitude to for providing the necessary facilities and support to carry out this research. We also extend our thanks to for their financial support under grant number. Special thanks to our collaborators for their valuable inputs and technical assistance. We appreciate the efforts of the laboratory staff and fellow researchers at for their constant support throughout this study.

REFERENCES

1. Prabhakaran, C., Kumar, P., Panneerselvam, N., Rajesh, S., & Shanmugam, G. (1996). Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells. Mutagenesis, 11(6), 553–557. https://doi.org/10.1093/mutage/11.6.553
2. Kumar, C. P., Pande, G., & Shanmugam, G. (1998). Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells. Apoptosis, 3(6), 413–419. https://doi.org/10.1023/a:1009658518998
3. Jearawuttanakul, K., et al. (2020). Cleistanthin A induces apoptosis and suppresses motility of colorectal cancer cells. European Journal of Pharmacology, 889, 173604. https://doi.org/10.1016/j.ejphar.2020.173604
4. Zhao, Y., et al. (2015). Synthesis and Evaluation of Cleistanthin A Derivatives as Potent Vacuolar H(+)-ATPase Inhibitors. Chemistry & Biodiversity, 12(10), 1583–1591. https://doi.org/10.1002/cbdv.201500112
5. Lu, Y., et al. (2016). ZT-25, a new vacuolar H(+)-ATPase inhibitor, induces apoptosis and protective autophagy through ROS generation in HepG2 cells. European Journal of Pharmacology, 771, 130–138. https://doi.org/10.1016/j.ejphar.2015.12.026
6. Whitton, B., et al. (2018). Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Cancer Medicine, 7(8), 3800–3811. https://doi.org/10.1002/cam4.1594
7. Pradheepkumar, C. P., & Shanmugam, G. (1999). Anticancer potential of cleistanthin A isolated from the tropical plant Cleistanthus collinus. Oncology Research, 11(5), 225–232.
8. Pan, S., et al. (2017). Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase-2 and -9. Oncology Letters, 14(5), 6217–6223. https://doi.org/10.3892/ol.2017.6917
9. Liu, S., et al. (2020). Cleistanthin A inhibits the invasion of MDA-MB-231 human breast cancer cells: involvement of the β-catenin pathway. Pharmacological Reports, 72(1), 188–198. https://doi.org/10.1007/s43440-019-00012-1
10. Ghosh, S. (2019). Cisplatin: The first metal based anticancer drug. Bioorganic Chemistry, 88, 102925. https://doi.org/10.1016/j.bioorg.2019.102925
11. Bray, F., et al. (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 68(6), 394–424. https://doi.org/10.3322/caac.21492
12. Demirci, N. S., et al. (2017). Modified docetaxel, cisplatin and fluorouracil therapy as the first-line treatment for patients with recurrent/metastatic squamous cell carcinoma of the head and neck cancer: a retrospective study. Current Medical Research and Opinion, 33(2), 401–407. https://doi.org/10.1080/03007995.2016.1257984
13. Parasuraman, S., & Raveendran, R. (2012). Diuretic effects of Cleistanthin A and Cleistanthin B from the leaves of Cleistanthus Collinus in Wistar Rats. Journal of Young Pharmacists, 4(2), 73–77. https://doi.org/10.4103/0975-1483.96616
14. Tuchinda, P., et al. (2006). Cytotoxic Arylnaphthalide Lignan Glycosides from the Aerial Parts of Phyllanthus taxodiifolius. Planta Medica, 72(1), 60–62. https://doi.org/10.1055/s-2005-873141
15. González, A. G., et al. (1979). Cytostatic activity of diphyllin and its derivatives. Planta Medica, 36(4), 345–349. https://doi.org/10.1055/s-0028-1097191
16. Lee, Y., & Kim, H. (2021). Overcoming chemoresistance in colorectal cancer: The role of natural compounds. Journal of Oncology, 2021.