br Conclusion Mar C selectively inhibited tumor
Conclusion: Mar-C selectively inhibited tumor growth via the induction of cancer cell senescence and had little chemotherapeutic toxicity, suggesting the potential of Mar-C as a promising anticancer agent.
General significance: This study provided evidence to identify a novelty of Mar-C that exerted antitumor activity on lung cancer through induction of senescence with limited toxicity.
Cellular senescence is an irreversible state of proliferative arrest while ML-210 still remain metabolically active. It is therefore believed that senescence is one of the tumor suppression mechanisms . In addition to “replicative senescence” with the shortening of telomere, telomere-independent cellular senescence could be induced by DNA damage, oxidative stress or oncogene activation, via diverse stimuli, termed as stress-induced premature senescence (SIPS) . In addition, premature senescence can be induced in cancer cells after exposed to many anti-tumor agents . For example, arsenite at low concentrations induced cellular senescence of human malignant glioblastoma cells instead of
apoptosis [4,5]. Human hepatoma cells displayed a senescence response exposed to a low concentration of chemotherapeutic doxorubicin, while they underwent apoptosis at a high concentration . Thus, cellular senescence has been regarded as a potent strategy for cancer therapy because of non-proliferative features of cells under the treatment at low concentrations of chemotherapeutic agents. Senescence is typically associated with a senescence-associated se-cretory phenotype (SASP), which contains the secreted inflammatory cytokines, proteases, chemokines and growth factors, and contributes to tumor suppression with elimination of senescent cells by triggering immune cells [7,8]. However, the paracrine effects of SASP changes the microenvironment of neighboring cells and tissues, leading to
Corresponding author at: Institute of Medical Sciences, The Second Hospital of Shandong University, 247 Beiyuan Street, Jinan 250033, China.
1 First author.
tumorigenesis and other pathological processes which is obstacle in senescence-induction therapy . Therefore, induction of cellular se-nescence with minimally increased SASP would be benefit for cancer chemotherapy . Marchantin C (Mar-C, Fig. 1A), a naturally-occurring product, was extracted from liverworts Marchantia polymorpha . As a member of macrocyclic bisbibenzyls, Mar-C is drawing our attention because it functioned as a microtubule inhibitor to induce apoptosis of human glioblastoma cells A172 and cervical carcinoma cells HeLa with an IC50 value ~8 μM and low toxicity [12,13]. In this study, we extended our investigations to the induction of cancer cellular senescence with the low concentrations of Mar-C below its IC50 and the amelioration on the promoting effects of SASP on tumor cell proliferation as genotoxic chemotherapies did.
2. Materials and methods
The human lung cancer cells A549, H460, H446, H1688 were pur-chased from ATCC and cultured in RPMI 1640 medium (HyClone) with 10% FBS (HyClone). The NSCLC H1299 cell line was a gift from Prof. Shi yikang (Shandong University) and maintained in DMEM medium (HyClone). The H460/RT cell line was established in our institute, by inducing H460 cell line with taxol. The normal human fibroblast NHF was cultured in DMEM medium with 10% FBS (Gibco). The human diploid cell strain IMR90 was purchased from Stem Cell Bank, Chinese Academy of Sciences, and maintained in MEM medium (Gibco) con-taining 10% FBS (Gibco), Gluta-max (2 mM, 3505061, Invitrogen), NEAA (100×, 1114050, Invitrogen), sodium pyruvate (1 mM, 11360070, Invitrogen). After cell confluence reached 50%–80%, the cells were treated with Mar-C as indicated. Dimethyl sulfoxide (DMSO) was used as the vehicle control.
2.2. Reagents and antibodies
Compound Mar-C  was dissolved in DMSO in a stock solution of
Cells were grown to 50%–60% confluence in a 6-well plate before transfection. Transfection of A549 cells was accomplished by adding Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to the siRNA di-lution. siRNA target sequences were synthesized from Genepharma (Shanghai, China) as follows: p21 siRNA sequences, 5′-GAU GUC CGU CAG AAC CCA UGCGGCA-3′; p65 siRNA sequences, 5′-GAU UGA GGA GAA ACG UAA ATT-3′.
2.4. SA-β-gal assay, clone formation assay and EdU assay
senescence in vitro was detected with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Boston, MA, USA). The pro-liferation ability of the lung cancer cells was analyzed with flat plate cloning experiments. The cells were seeded with 600 cells·mL−1 in a 6-well plate. The culturing was lasted for 10 d until the clones could be observed with naked eyes (> 50 cells/clone). The clones were then fixed and stained with Giemsa's Stain Kit (Solarbio, China). Colonies were counted and measured with images.