Anti-Proliferative and Pro-Apoptotic Effects Of Dipsacus Asperoides in a Cellular Model for Triple-Negative Breast Cancer

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Nitin Telang
Hareesh Nair
George YC Wong


Breast cancer, Dipsacus asperoides, Pro-apoptotic, TNBC


Background: Triple negative breast cancer (TNBC) lacks expressions of estrogen receptor-α (ER-α), progesterone receptor (PR) and amplified human epidermal growth factor receptor-2 (HER-2). Current treatment for TNBC includes anthracyclin, taxol and cisplatin-based conventional chemotherapy and survival pathway PARP, PI3K, AKT and mTOR selective targeted therapy. These treatments exhibit dose-limiting systemic toxicity and presence of drug resistant cancer stem cells, which highlight the need for identification of efficacious testable alternatives that are not toxic to non-tumorigenic cells. Dipsacus asperoides (DA) is a Chinese nutritional herb and its root represents a common ingredient in Chinese herbal formulations used in women for estrogen related health issues, osteoporosis and breast diseases. This study aims to investigate the growth inhibitory effects of DA, and to detect mechanisms for its efficacy.
Methods: Human mammary carcinoma derived triple negative MDA-MB-231 cell line represented the TNBC model. Non-fractionated aqueous extract from DA represented the test agent. Anchorage dependent growth, anchorage independent (AI) colony formation and cell cycle progression quantified growth inhibition. Western blot-based analysis for inhibition of RAS, PI3K and AKT and RB signaling identified mechanistic leads.
Results: Treatment with DA induced a dose dependent cytostatic growth arrest (IC50:15 µg/ml; IC90: 30 µg/ml), reduced AI growth and inhibited cell cycle progression via G2/M arrest. DA affected the RAS, PI3K, AKT and RB signaling pathways, and functioned as a natural inhibitor of cyclin dependent kinase 4/6. Cellular apoptosis paralleled increase in pro-apoptotic Caspase 3/7 activity.  
Conclusion: These results demonstrate that DA inhibited growth, affected cell cycle progression, induced apoptosis and inhibited cancer cell survival pathways. This study validates a mechanism-based approach to identifying testable substitutes for secondary prevention/therapy of TNBC.


1. American Cancer Society-Facts & Figures 2021. Atlanta: American Cancer Society, 2021.
2. Sorlie T, Perou CM, Tibshirany R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc. Natl. Acad. Sci. USA. 2001; 98: 10869-10874. doi: 10.1073/pnas.191367098.
3. Baselga J, Swain SM. Novel anti-cancer agents: Revisiting ERBB2 and discovering ERBB3. Nat. Rev. Cancer. 2009; 9: 463-475. doi: 10.1038/nrc 2656.
4. Dinh P, Satirou C, Piccart MJ. The evaluation of treatment strategies: Aiming at the target. Breast. 2007; 16 (suppl. 2): S10-S16. doi: 10.1016/j. breast. 2007.07.032
5. Anders CK, Winer EP, Ford JM, Dent R, Silver DP, Sledge GW, et al. Poly (ADP-ribose) inhibition: Targeted therapy for triple negative breast cancer. Clin. Cancer Res. 2010; 16: 4702-4710. doi: 10.1158/1078-0432.CCR-10-0939.
6. Dean M, Fojo T, Bates S. 2005; Tumor stem cells and drug resistance. Nat. Rev. Cancer. 2005; 5: 275-284. doi: 10.1038/nrc 1590.
7. Tindle HA, Davis RB, Phillips RL, Eisenburg DM. Trends in the use of complementary and alternative medicines by US adults: 1997-2002. Altern. Ther. Health Med. 2005; 11: 42-49. doi: Not available.
8. Molassiotis A, Scott JA, Kearney A, Kearney N. Complementary and alternative medicine use in breast cancer patients in Europe. Support Care Cancer. 2006; 14: 260-267. doi: 10.1007/sco520-005-0883-7.
9. Hyler LK, Chin S, Chui BK: The use of complementary and alternative medicine among patients with locally advanced breast cancer: A descriptive study. BMC Cancer. 2006; 6: 39-46. doi: Not available.
10. Ye L, Jia Y, Ji KE, Saunders AJ, Xue K, Ji J, et al. Traditional Chinese medicine in the prevention and treatment of breast cancer and cancer metastasis. Oncol. Lett. 2015; 10: 1240-1250. doi: 10.3892/ol.2015.3459.
11. Mukherjee B, Telang N, Wong GYC. Growth inhibition of estrogen receptor positive human breast cancer cells by Taheebo from the inner bark of Tabebuia avellandae tree. Int. J. Mol. Med. 2009; 24: 253-260. doi: 10.3892/ijmm.00000228.
12. Li G, Sepkovic DW, Bradlow HL, Telang NT, Wong GYC: Lycium barbarum inhibits growth of estrogen receptor positive human breast cancer cells by favorably altering estradiol metabolism. Nutrition & Cancer. 2009; 61: 408-414. doi: 10.1080/0163558082585952.
13. Telang NT, Li G, Sepkovic DW, Bradlow HL, Wong GYC. Anti-proliferative effects of Chinese herb Cornus officinalis in a cell culture model for estrogen receptor positive clinical breast cancer. Mol. Med. Rep. 2012; 5: 22-28. doi: 10.3892/mmr.2011.617.
14. Telang N, Li G, Sepkovic D, Bradlow HL, Wong GYC. Comparative efficacy of extracts form Lycium barbarum bark and fruit on estrogen receptor positive human mammary carcinoma MCF-7 cells. Nutrition & Cancer. 2014; 66: 278-284. doi: 10.1080/01635581.2014.864776.
15. Telang N, Li G, Katdare M, Sepkovic D, Bradlow L, Wong GYC. Inhibitory effects of Chinese nutritional herbs in isogenic breast carcinoma cells with modulated estrogen receptor function. Oncol. Lett. 2016; 12: 3949-3957. doi: 10.3892/ol.2016.5197.
16. Telang NT, Nair HB, Wong GYC. Growth inhibitory efficacy of Tabebuia avellanedae in a model for triple negative breast cancer. Arch. Breast Cancer 2021; 8: 203-209. doi: 10.32768/abc.202183203-209.
17. Telang N, Nair HB, Wong GYC. Growth inhibitory efficacy of Cornus officinalis in a cell culture model for triple negative breast cancer. Oncol. Letts. 2019; 17: 5261-5266. doi: 10.3892/ol.2019.10182.
18. Neve RM, Chin K, Fridyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006; 10: 515-527. doi: 10.1016/jccr.2006.10.008.
19. Subik K, Lee J-F, Baxter L, Strzepak T, Costello D, Crowley P, et al. Expression patterns of ER, PR, HER-2, CK5/6, EGFR, Ki67 and AR by immuono-histochemical analysis in breast cancer cell lines. Breast Cancer (Aukl.) 2010; 4: 35-41. doi: Not available.
20. Hudis CA, Gianni L. Triple negative breast cancer: An unmet medical need. Oncologist Suppl. 2011; 1: 1-11. doi: 10.1634/theoncologist.2011-s1-01.
21. Lin NU, Vanderplast A, Hughes ME, Theriault RL, Edge SB, Wong U-N, et al. Clinico-pathologic features, patterns of recurrence and survival among women with triple negative breast cancer in the National Comprehensive Cancer Network. Cancer. 2012; 218: 5463-5472. doi: 10.1002/cncr.27581.
22. Telang N. Putative cancer initiating stem cells in cell culture models for molecular subtypes of clinical breast cancer. 2015; Oncol. Lett. 10: 3840-3846. doi: 10.3892/ol.2015.3780.
23. Cortes J, Baselga J. Targetting the microtubules in breast cancer beyond taxanes: the epothilones. Oncologist. 2007; 12: 271-280. doi: 10.1634/theoncologist.12-3-271.
24. Thomas G, Sreeja JS, Gireesh KK, Gupta H, Manna TK. +TIP EB1 downregulates paclitaxel-induced proliferation inhibition and apoptosis in breast cancer cells through inhibition of paclitaxel binding to microtubules. Int. J. Oncol. 2015; 46: 133-146. doi: 10.3892/ijo.2014.2701.
25. Mirzoeva OK, Das D, Heiser LM, Bhattacharya S, Sivak D, Gendelman R, et al. Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition. Cancer Res. 2009; 69: 565-572. doi: 10.1158/0008-5472.CAN-08-3389.
26. Collisson EA, Trejo CL, Silvo JM, Gu S, Karkola JE, Heiser LM, et al. A central role of RAF-MEK-ERK signaling in genesis of pancreatic ductal adenocarcinoma. Cancer Discov. 2012; 2: 685-693. doi: 10.1158/2159-8290.CD-11-0347.
27. Nussinov R, Tsai C-J, Jang H. Oncogenic Ras isoforms signaling specificity at the membrane. Cancer Res. 2018; 78: 593-602. doi: 10.1158/0008-5472.CAN-17-2727.
28. Yaeger R, Solit DB. Overcoming adaptive resistance to KRAS inhibitors through vertical pathway targeting. Clin. Cancer Res. 2020; 26: 1538-1540. doi: 10.1158/1078-0432.CCR-19-4060.
29. Ryan MB, Face de la Cruz F, Phat S, Meyers DT, Wong E, Shahzade HA, et al. Vertical pathway inhibition overcomes adaptive feedback resistance to KRAS G12c inhibition. Clin. Cancer Res. doi; Not available. PMID: 31776128.
30. Gohr K, Hamacher A, Engelke LH, Kassak MU. Inhibition of PI3K/AKT/mTOR overcomes cisplatin resistance in the triple negative breast cancer cell line HCC38. BMC-Cancer. 2017; 17: 711. doi: 10.1186/s 12885-017-3695-5.
31. Massihnia D, Galvano A, Fanale D, Perez A, Casiglia M, Incorvaia L, et al. Triple negative breast cancer: Shedding light on to the role of PI3K/AKT/mTOR pathway. Oncotarget. 2016; 7: 60712-60722. doi: 10.18632/oncotarget.10858.
32. Costa RLB, Han HS, Gradishar WJ. Targeting the PI3K/AKT/mTOR pathway in triple negative breast cancer: A review. Breast Cancer Res. Treat. 2018; 169: 397-406. doi: 10.1007/s10549-018-4697-Y.
33. Jaglanian A, Tsiani E. Rosemary extract inhibits proliferation, survival, AKT, and mTOR signaling in the triple negative breast cancer cells. Int. J. Mol. Sci. 2020; 21: 810. doi: 10.3390/ijms21030810.
34. Memmott RM, Dennis PA. Akt-dependent and-independent mechanisms of mTOR regulation in cancer. Cell Signal. 2009; 21: 656-664. doi: 10.1016/j.cellsig.2009.01.004.
35. Jegg AM, Ward TM, Iorns E, Hoe N, Zhou J, Liu X, et al. PI3K independent activation of mTORC1 as a target in lapatinib-resistant ERBB2 + breast cancer cells. Breast Cancer Res. Treat. 2012; 136: 683-692. doi: 10.1007/s10549-012-2252-9.
36. Cox LA, Chen G, Lee EY. Tumor suppressor genes and their role in breast cancer. Breast Cancer Res. Treat. 1994; 32: 19-38. doi: 10.1007/BF00666203.
37. Burkhart DL, Sage J. Cellular mechanisms of tumor suppression by the retinoblastoma gene. Nat. Rev. Cancer. 2008; 8: 671-682. doi: 10.1038/nrc 2399.
38. Bosco EE, Knudson ES. RB in breast cancer: At the crossroads of tumorigenesis and treatment. Cell Cycle. 2007; 6: 667-671. doi: 10.4161/cc.6.6.3988.
39. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1 phase progression. Genes & Dev. 1999; 13: 1501-1512. doi: 10.1101/gad.13.12.1501.
40. Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL. Cyclin D as a therapeutic target in cancer. Nat. Rev. Cancer. 2011; 11: 558-572. doi: 10.1038/nrc 3090.
41. Van Arsdale T, Boschoff C, Arndt KT, Abraham RT. Molecular pathways: Targeting the Cyclin D-CDK 4/6 axis for cancer treatment. Clin. Cancer Res. 2015; 21: 2905-2910. doi: 10.1158/1078-0432.CCR-14-0816.
42. Telang N, Nair HB, Wong, GYC. Efficacy of Dipsacus asperoides (DA) in a model for triple negative breast cancer. Cancer Res. 2016; 76 (Suppl.): SABCS Abstract # P4-13-04. doi: not available.
43. Lee HL, Lin CS, Kao SH, Chou MC. Gallic acid induces G1phase arrest and apoptosis of triple negative breast cancer cell MDA-MB-231 via p38 mitogen-activated protein kinase /p21/p27 axis. Anticancer Drugs. 2017; 28: 1150-1156. doi: 10.1097/CAD.0000000000000565.
44. Giordano C, Rovito D, Barone I, Moncuso R, Bonofiglio D, Giordano F, et al. Benzofuran-2-acetic ester derivatives induce apoptosis in breast cancer cells by upregulating p21Cip/WAF1 gene expression in p53-independent manner. DNA Repair. 2017; (Amst.) 51: 20-30. doi: 10.1016/jdnarep.2017.01.006.
45. Muller PAJ, Vousden KH. Mutant p53 in cancer: New functions and therapeutic opportunities. Cancer Cell. 2014; 25: 304-317. doi: 10.1016/j.ccr.2014.01.021.
46. Hui L, Zheng Y, Yan Y, Bargonetti J, Foster DA. Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D. Oncogene. 2006; 25: 7305-7310. doi: 10.1038/sj.onc.1209735.
47. Choi YH, Kang HS, Yoo MA. Suppression of human prostate cancer cell growth by β-Lapachone via down-regularion of pRB phosphorylation and induction of CDK inhibitor p21 (WAF1/CIP1). J. Biochem. Mol. Biol. 2003; 36: 223-229. doi: 10.5483/bmbrcp.2003.36.2.223.
48. Boer K. Impact of palbociclib combinations on treatment of advanced estrogen receptor-positive/human epidermal growth factor receptor2-negative breast cancer. Onco Targets Ther. 2016; 11: 6119-6125. doi: 10.2147/OTT.s77033.
49. Alves CL, Elias D, Lyng M, Bak M, Kirkegaard T, Lykkesfeldt AE et al. High CDK6 protects cells from Fulvestrant-mediated apoptosis and is a predictor of resistance to Fulvestrant in estrogen receptor-positive metastatic breast cancer. Clin. Cancer Res. 2016; 22: 5514-5526. doi: 10.1158/1078-0432.CCR-15-1984.
50. Klein ME, Kovatcheva M, Davis LE, Tap WD, Koff A. CDK4/6 inhibitors: The mechanism of action may not be as simple as once thought. Cancer Cell. 2018; 34: 9-20. doi: 10.1016/j.ccell.2018.03.023.
51. Asghar US, Barr AR, Cutts R, Beany M, Babina I, Sampath D, et al. Single cell dynamics determines response to CDK4/6 inhibition in triple negative breast cancer. Clin. Cancer Res. 2017; 23: 5561-5572. doi: 10.1158/1078-0432.CCR-17-0369.
52. Foidart P, Yip C, Redenmacher J, Blacher S Lienard M, Montero-Ruiz L, et al. Expression of MT4-MMP, EGFR and RB in triple negative breast cancer strongly sensitizes tumors to erlotinib and palbociclib combination therapy. Clin. Cancer Res. 2019; 25: 1838-1850. doi: 10.1158/1078-0432.CCR-18-1880.
53. Li T, Xiong Y, Wang Q, Chen F, Zeng Y, Yu X, et al. Ribociclib (LEE011) suppresses cell proliferation and induces apoptosis of MDA-MB-231 cells by inhibiting CDK4/6-cyclin D-Rb-E2F pathway. Artif. Cells Nanomed. Biotechnol. 2019; 47: 4001-4011. doi: 10.1080/21691401.2019.1670670.
54. Teo ZL, Versaci S, Dushyanthen S, Caramia F, Savas P, Mintoff CP, et al. Combined CDK4/6 and PI3Kα inhibition is synergistic and immunogenic in triple-negative breast cancer. Cancer Res. 2017; 77: 6340-6352. doi: 10.1158/0008-5472.CAN-17-2210.
55. De Leeuw R, McNair C, Schiewer MJ, Neupane NP, Brand LJ, Augello MA, et al: MAPK reliance via acquired CDK4/6 inhibitor resistance in cancer. Clin. Cancer Res. 2018; 24: 4201-4214. doi: 10.1158/1078-0432. CCR-18-0410.

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