Effect of CP metronomic chemotherapy on HUVEC cells and autophagy-related protein Beclin1 and LC3
GUO Lieping1, ZHOU Fan1, SHI Haotian1, CHEN Haimin1, LIN Chenhui1, CHEN Xiaoling1, HOU Jian1, 2
1. Department of Hematology and Oncology, Shanghai Zhabei District Central Hospital, Shanghai200070, China; 2. Department of Hematology, Changzheng Hospital, the Second Military Medical University, Shanghai 200003, China
Abstract：Objective To investigate the effect of CP metronomic chemotherapy on HUVEC cells and autophagy-related protein, Beclin1 and LC3, expression levels, and to explore the mechanism of CP program increasing sensitivity of HUVEC cells to chemotherapeutic drug. Methods Subjects were divided into the DMSO control group, and the phosphoramide mustard (PM) group, the prednisolone group, the phosphoramide mustard and prednisolone group (the CP group). HUVEC cells were treated with different drugs. CCK8 method was used to detect cell proliferation, flow cytometry to detect the cell apoptosis, Western blot and RT-PCR to detect autophagy-related protein levels. Results CCK8 method showed that compared with DMSO control group, in the PM, the prednisolone and the CP groups, HUVEC cell proliferation inhibition rates increased significantly with the extension of time (P<0.001). And at the same time, the different drug groups were significantly different in cell proliferation inhibition rate, the prednisone group was the weakest, the CP group(72 h,25.1%±0. 7%)was the strongest, and the PM group (72 h,20.4%±0.7%) was intermediate(P<0.01). Flow cytometry showed that after 48 hours, compared with the DMSO control group, the prednisolone, the PM and the CP groups made HUVEC cell apoptosis rate increased in turn (P<0.001). Western blot and reverse transcription PCR showed that after 48 hours, compared with the DMSO control group, Beclin1 and LC3 protein and mRNA expression decreased in turn in the prednisolone group, the PM group and the CP group(P<0.001). Conclusions HUVEC cells treated with CP metronomic chemotherapy can significantly reduce the level of HUVEC cell proliferation, promote HUVEC cell apoptosis, and significantly reduce autophagy-related protein expression levels of Beclin1 and LC3.
Suvannasankha A, Fausel C, Juliar B E, et al. Final report of toxicity and efficacy of a phase II study of oral cyclophosphamide, thalidomide, and prednisone for patients with relapsed or refractory multiple myeloma: a Hoosier Oncology Group Trial,HEM01-21[J]. Oncologist, 2007, 12(1): 99-106.
[19]
Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism [J]. Oncogene, 2004, 23(16): 2891-2906.
[20]
Holm E, Hildebrandt W, Kinsecherf R. Low postabsorptive net prtein degradation in male cancer patients: lack of sensitivity to regulatory amino acids [J]. Oncol Rep, 2007, 17(3): 695-700.
Satoh J, Motohashi N, Kino Y, et al. LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains [J]. Orphanet J Rare Dis, 2014, 9: 68.
Zhou F, Guo L, Shi H, et al. Continuous administration of low-dose cyclophosphamide and prednisone as a salvage treatment for multiple myeloma [J]. Clin Lymphoma Myeloma Leuk, 2010, 10(1): 51-55.
[2]
Zhou F, Ling C, Guo L, et al. Continuous low-dose cyclophosphamide and prednisone in the treatment of relapsed/refractory multiple myeloma with severe heart failure [J]. Leuk Lymphom, 2014, 55(10): 2271-2276.
[24]
Sun Y, Liu J H, Sui Y X, et al. Beclin1 overexpression inhibitis proliferation, invasion and migration of CaSki cervical cancer cells [J]. Asian Pac J Cancer Prev, 2011, 12(5): 1269-1273.
[25]
Guo X L, Li D, Sun K, et al. Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma [J]. J Mol Med (Berl), 2013, 91(4): 473-483.
Nicolini A, Mancini P, Ferrari P, et al. Oral low-dose cyclophosphamide in metastatic hormone refractory prostate cancer (MHRPC)[J]. Biomed Pharmacother, 2004, 58(8): 447-450.
[9]
Bocci G, Tuccori M, Emmenegger U, et al. Cyclophosphamide- methotrexate 'metronomic' chemotherapy for the palliative treatment of metastatic breast cancer. A comparative pharmacoeconomic evaluation [J]. Ann Oncol, 2005, 16(8): 1243-1252.
[10]
Munoz R, Shaked Y, Bertolini F, et al. Anti-angiogenic treatment of breast cancer using metronomic low-dose chemotherapy[J]. Breast, 2005, 14(6): 466-479.
[11]
Baruchel S, Diezi M, Hargrave D, et al. Safety and pharmacokinetics of temozolomide using a dose-escalation, metronomic schedule in recurrent paediatric brain tumours[J]. Eur J Cancer, 2006, 42(14): 2335-2342.
[12]
Kong D S, Lee J I, Kim W S, et al. A pilot study of metronomic temozolomide treatment in patients with recurrent temozolomide-refractory glioblastoma [J]. Oncol Rep, 2006, 16(5): 1117-1121.
[13]
Stempak D, Gammon J, Halton J, et al. A pilot pharmacokinetic and antiangiogenic biomarker study of celecoxib and low-dose metronomic vinblastine or cyclophosphamide in pediatric recurrent solid tumors [J]. J Pediatr Hematol Oncol, 2006, 28(11): 720-728.
[14]
Young S D, Whissell M, Noble J C, et al. Phase II clinical trial results involving treatment with low-dose daily oral cyclophosphamide, weekly vinblastine, and rofecoxib in patients with advanced solid tumors [J]. Clin Cancer Res, 2006, 12(10): 3092-3098.
[15]
Coleman M, Martin P, Ruan J, el al. Low-dose metronomic, multidrug therapy with the PEP-C oral combination chemotherapy regimen for mantle cell lymphoma [J]. Leuk Lymphoma, 2008, 49(3): 447-450.
[16]
De Weerdt O, Vande Donk N W, Veth G, et al. Continuous low-dose cyclophosphamide-prednisone is effective and well tolerated in patients with advanced multiple myeloma [J]. Neth J Med, 2001, 59(2): 50-56.
[17]
Trieu Y, Trudel S, Pond G R, et al. Weekly cyclophosphamide and alternate-day prednisone: an effective, convenient, and well-tolerated oral treatment for relapsed multiple myeloma after autologous stem cell transplantation [J]. Mayo Clin Proc, 2005, 80(12): 1578-1582.
[18]
Suvannasankha A, Fausel C, Juliar B E, et al. Final report of toxicity and efficacy of a phase II study of oral cyclophosphamide, thalidomide, and prednisone for patients with relapsed or refractory multiple myeloma: a Hoosier Oncology Group Trial,HEM01-21[J]. Oncologist, 2007, 12(1): 99-106.
[19]
Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism [J]. Oncogene, 2004, 23(16): 2891-2906.
[20]
Holm E, Hildebrandt W, Kinsecherf R. Low postabsorptive net prtein degradation in male cancer patients: lack of sensitivity to regulatory amino acids [J]. Oncol Rep, 2007, 17(3): 695-700.
Satoh J, Motohashi N, Kino Y, et al. LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains [J]. Orphanet J Rare Dis, 2014, 9: 68.
Sun Y, Liu J H, Sui Y X, et al. Beclin1 overexpression inhibitis proliferation, invasion and migration of CaSki cervical cancer cells [J]. Asian Pac J Cancer Prev, 2011, 12(5): 1269-1273.
[25]
Guo X L, Li D, Sun K, et al. Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma [J]. J Mol Med (Berl), 2013, 91(4): 473-483.
2015, Vol. 26 
