Cotylenin Α, a Fusicoccane Diterpene Glycoside with a Complex Sugar Moiety, and Vincristine Synergistically Inhibit the Growth of Myeloma Cell in Vitro and in Vivo
Authors: Takahashi Tsutomu , Honma Yoshio , Kawakami Koshi , Suzumiya Junji 
Published: October 2019

Abstract
Introduction: Multiple myeloma is still incurable and optimize existing chemotherapeutic strategies and development of novel agents are necessary to improve the outcome of patients. Cotylenin A, a fusicoccane diterpene glycoside with a complex sugar moiety, was isolated as a plant-growth regulator. Cotylenin A modulates the 14-3-3 intracellular signaling pathway and has been shown to inhibit the growth of several cancer cells. Herein, we examined the antitumor effects of cotylenin A to develop a novel treatment against myeloma.
Methods: Five human myeloma cell lines, RPMI8226, KMS-11, KMS-26, KMS-12 PE and KMS-12 BM were cultured with cotylenin A alone or in combination with several anticancer drugs. To measure the effects of various drugs on the growth of myeloma cells, the number of viable cells was determined by the MTT assay after 6 days of exposure to various concentrations of drugs with or without 2 μg/ml cotylenin A. The growth-inhibiting effects of the drugs were examined by determining the concentrations of drugs required to reduce the cell number to one-half of that in untreated cells (IC50). Xenografts in six-week-old female (Fox Chase SCID C.B-17/Icr-scid Jcl) mice were used to determine the in vivoefficacy of cotylenin A.
Results: Cotylenin A alone inhibited the growth of myeloma cells in dose dependent manner, but the effect of growth inhibition was relatively weak. Cotylenin A and vincristine synergistically inhibited the growth and induced apoptosis in myeloma cells. While other microtubule-disturbing agents also showed co-operative effects with cotylenin A, other anticancer agents, such as doxorubicin, cisplatin, camptothecin, methotrexate, gemcitabine and 5-fluorouracil, did not show such co-operation with cotylenin A (Table 1). Cotylenin A had synergistic effects with vincristine and the results were confirmed by an isobologram analysis. These differences might be attributed to the effects on autophagic responses. Combined treatment with cotylenin A and vincristine induced autophagy (formation of LC3-II and degradation of p62 protein). However, doxorubicin did not enhance the autophagy induced by cotylenin A. The induction of apoptosis was confirmed by an analysis of the DNA histogram and the expression of annexin V. The expression of cleaved caspase-3 was increased in treatment with 2 ng/ml vincristine and enhanced by 2 μg/ml cotylenin A by western blot analysis. An invasion assay using transwell chamber revealed that low concentration cotylenin A (0.6 μg/ml) effectively inhibited the invasive activity of RPMI 8226 myeloma cells without inhibiting growth. A colony-forming assay indicated that 2 μg/ml cotylenin A preferentially inhibited the formation of large colonies, which have high self-renewal activity. The combined treatment with 3 μg/ml cotylenin A and 3 ng/ml vincristine more effectively suppressed the formation of large colonies than vincristine alone. Expression of pluripotency-associated transcription factor Sox2 mRNA in RPMI 8226 myeloma cells was significantly suppressed by treatment with 4 μg/ml cotylenin A. Combined treatment with 5 mg/kg cotylenin A and 0.5 mg/kg vincristine significantly inhibited the growth of KMS-26 myeloma cells as xenografts. In addition, mice with cotylenin A and vincristine showed the reduction of body weight loss comparing with those of mice by vincristine alone. This result supported that the combination of cotylenin A and vincristine might be safe.
Conclusions: Cotylenin A and vincristine synergistically inhibited the growth of myeloma cells in vitro and in vivo, and the invasion of myeloma cells. Our results suggest that the combination of cotylenin A and vincristine may have therapeutic value for myeloma.
Table 1. Potentiation of the growth-inhibitory activities of various anticancer agents in RPMI8226 myeloma cells by cotylenin A. Growth inhibition (IC50) Anticancer agent (ng/ml) - Cotylenin A + Cotylenin A Ratio(-/+) Doxorubicin 5.5 ± 0.6 5.3 ± 0.5 1.03 Cisplatin 246 ± 30.2 237 ± 28.4 1.03 Camptotecin 1.42 ± 0.16 1.38 ± 0.14 1.03 Methotrexate 2.76 ± 0.3 1.81 ± 0.2 1.52 Gemcitabine 4.3 ± 0.4 3.1 ± 0.3 1.39 5-Fluorouracil 56.5 ± 5.1 57.8 ± 6.3 0.98 Vincristine 6.3 ± 0.6 1.6 ± 0.1 3.94 Vinblastine 0.91 ± 0.11 0.42 ± 0.05 2.17 Paclitaxel 16.4 ± 1.9 7.7 ± 0.9 2.13
Ratio (-/+), IC50 without cotylenin A: IC50 with cotylenin A. The values are the mean ± SD of four determinations.

Disclosures
No relevant conflicts of interest to declare.