Continuous low-dose administration of GEM and/or CDDP facilitated tumor formation and promoted tumor growth in vivo
Surprisingly, we found that continuous low-dose administration of GEM and GEM + CDDP significantly promoted tumor growth in xenograft tumor models in vivo.
As shown in Fig. 1a-c, in a B16 tumor model, once every 48h administration of 1.25 mg/kg GEM and 0.6/0.03 mg/kg GEM + CDDP for 2 weeks remarkably induced in B16 tumor growth by 45.2% and 32.2%, respectively, relative to control (each p < 0.05). In contrast, once every 48h administration of 5 mg/kg GEM, 2.4/0.12 and 4.8/0.24 mg/kg GEM + CDDP for 2 weeks markedly inhibited tumor growth. Of note, no significant effect was found in tumor treated with CDDP alone. In addition, the average tumor weights in the 5 mg/kg GEM and 4.8/0.24 mg/kg GEM + CDDP groups after 2 weeks treatment were reduced by 36.6% and 39.1%, respectively, relative to control (both p < 0.05). Strikingly, in MCF-7 mediated nude mouse tumor model, out of six tested mice, we found some tumors (from 1 to 4) developed from either GEM or GEM + CDDP low-dose treatment groups but not in the control group 6 weeks after implanting equal number of MCF-7 tumor cells (Fig. 1d). Together these findings indicate that the continuous low-dose administration of GEM and GEM + CDDP can promote significant tumor formation and growth in vivo.
Low-concentration GEM and GEM + CDDP promoted B16, MCF-7, and T-47D cell functions in vitro
Based on the surprising in vivo findings, we intended to test the effects of GEM and CDDP on the proliferation of B16 cells, MCF-7 and T-47D tumor cells. The cell proliferation was measured by MTT assay. As shown in Fig. 2a, low-concentration GEM (1×10− 3 or 5×10− 5 µM) and GEM + CDDP treatment promoted the proliferation of MCF-7 cells, whereas 10 or 0.5 µM GEM and GEM + CDDP significantly suppressed cell proliferations (both p < 0.01). In contrast, no significant effect was found in CDDP-treated B16 or MCF-7 cell. These results implied that levels of antineoplastic agents used have a significant impact on the fate of tumor cells, e.g., proliferation or cell death.
Meanwhile, although the low concentrations of 1×10− 5µM GEM or 5×10− 7µM CDDP did not affect the proliferation of T-47D tumor cells alone, their combination at these concentrations resulted in marked facilitation (p < 0.05) (Fig. 2b). Similar phenomena were found in B16 cells with 1×10− 3 µM GEM and 5×10− 5 µM CDDP (note that these concentrations differ from those applied to T-47D cells) (p < 0.05) (Fig. 2c).
We also performed the colony formation and wound healing assays. As shown in Fig. 2d, colony formation was markedly enhanced in B16 cells at 1×10− 3 µM GEM and 1×10− 3/5×10− 5 µM GEM + CDDP groups relative to control (both p < 0.01). Similar results were seen in the wound-healing assay showing that low-concentration 1×10− 3 µM GEM or 1×10− 3/5×10− 5 µM GEM + CDDP promoted the proliferation of B16 cells (both p < 0.01) (Fig. 2e).
The effects of GEM or GEM + CDDP on the invasion and migration functions of B16, MCF-7 and T-47D cells were investigated at low concentrations. The results are shown in Fig. 2f-h, relative to control, the invasion and migration of tumor cells were significantly enhanced in MCF-7 cells after 1×10− 3 µM GEM and 1×10− 3/5×10− 5 µM GEM + CDDP treatment, in T-47D cells after 1×10− 5 µM GEM and 1×10− 5/5×10− 7 µM GEM + CDDP treatment, and in B16 cells after 1×10− 3 µM GEM and 1×10− 3/5×10− 5 µM GEM + CDDP treatment (each p < 0.01). These results indicate that both GEM and GEM + CDDP can enhance the tumor-cell functions at low-concentration in vitro.
In addition, the cell-cycle assay revealed that 1×10− 3 µM GEM and/or 5×10− 5 µM CDDP markedly decreased the fraction of MCF-7 and B16 cells at the G2 and M phases of the cell cycle, while 1×10− 5 µM GEM and/or 5×10− 7 µM CDDP decreased the fraction of T-47D cells at the G0 and G1 phases (each p < 0.01) (Fig. 2i-k).
Low-concentration GEM and GEM + CDDP inhibited apoptosis of B16, MCF-7, and T-47D cells in vitro
In a standard culture condition, we observed approximately 10–25% apoptotic cells in B16, MCF-7, and T-47D cells. Intriguingly, when we treated tumor cells with low-concentration of GEM and GEM + CDDP (1×10− 5 µM and 1×10− 5/5×10− 7 µM, respectively), the apoptotic cells were profoundly declined by 44.1% and 68.9%, respectively, relative to non-treated MCF-7 cells (both p < 0.01), by 41.1% and 55.4% in B16 cells (p < 0.01 and p < 0.05 respectively), and by 50.4% and 67.4% in T-47D cells (both p < 0.01) (Fig. 3a-c). In addition, MCF-7 cells after 1×10− 3 µM GEM and 1×10− 3/5×10− 5 µM GEM + CDDP treatment increased the antiapoptotic protein Bcl-2 by 92.1% and 136.2%, while decreased the proapoptotic Bax protein by 35.2% and 53.3%, respectively, compared to non-treated cells. Furthermore, caspase-3 expression levels in MCF-7 cells were markedly decreased by 29.8% and 68.6%, cleaved-caspase-3 were decreased by 34.9% and 54.5% after treatment with 1×10− 3 µM GEM and 1×10− 3/5×10− 5 µM GEM + CDDP, respectively, compared to non-treated cells. Also, caspase-9 expression levels were decreased by 48.1% and 76.2%, and cleaved-caspase-9 were decreased by 15.6% and 43.9%. Similarly results were seen in low-dose GEM and GEM + CDDP treated T-47D cells, 1×10− 5 µM GEM and 1×10− 5/5×10− 7 µM GEM + CDDP treatment increased Bcl-2 expression by 7.3% and 172.7%, respectively, and decreased Bax expression by 55.5% and 55.6%, caspase-3 expression by 17.2% and 20.3%, cleaved-caspase-3 expression by 41.2% and 63.4%, caspase-9 expression by 31.1% and 27.8% and cleaved-caspase-9 expression by 16.9% and 36.8%, respectively, compared to non-treated cells (Fig. 3d-e).
Those findings strongly suggest that the tumor growth acceleration induced by low-concentration GEM and GEM + CDDP is associated with inhibition of the expression of proapoptotic proteins and promotion of the expression of antiapoptotic proteins.
Low-dose GEM and GEM + CDDP promoted tumor angiogenesis in vivo, and inhibited the proliferation of endothelial cells in vitro
As shown in Fig. 4a-b, the areas of CD31+ vessels in 1.25 mg/kg GEM and 0.6/0.03 mg/kg GEM + CDDP groups were significantly enhanced in B16 tumor tissues relative to control (both p < 0.01), as was also the case in laminin positive vessels (both p < 0.01). This implies that GEM and GEM + CDDP can promote tumor angiogenesis under continuous low-dose administration conditions. However, we found that GEM and GEM + CDDP inhibited endothelial cells proliferation in the concentration range from 1×10− 2/5×10− 4 µM to 10/0.5 µM in vitro (each p < 0.01), with no stimulation role of GEM or GEM + CDDP found at lower concentrations (Fig. 4c).
These results suggest that GEM and GEM + CDDP can promote angiogenesis in B16 tumor tissues at low-dosage via pathways independent of endothelial cells stimulation.
Continuous low-dose administration of GEM and GEM + CDDP promoted the mobilization of Gr-1+CD11b+ and CD61+BMDCs
To further explore the potential mechanisms of low-dosage GEM and GEM + CDDP induced angiogenesis, we measured the proangiogenic BMDCs, e.g., Gr-1+CD11b+ and CD61+BMDCs. As shown in Fig. 5a, the proportions of Gr-1+CD11b+ BMDCs were increased by 52.3%, 53.1% and 17.7% in 1 mg/kg GEM, 4 mg/kg GEM and 0.2 mg/kg CDDP groups, respectively, compare to saline control group. The proportions of Gr-1+CD11b+ BMDCs were increased by 331.7% and 406.2% in the 1/0.05 and 4/0.2 mg/kg GEM + CDDP groups respectively, relative to control (both p < 0.01). Combination of GEM and CDDP significantly enhanced the mobilization of Gr-1+CD11b+BMDCS compared to GEM or CDDP alone (all p < 0.01). The Gr-1+CD11b+ and CD61+BMDCs counts were increased by 65.1% and 18.6%, respectively, in B16 tumor bearing C57BL/6J mice after 2 weeks treatment with 1.25 mg/kg GEM (both p < 0.05) (Fig. 5b-c). The average proportions of Gr-1+CD11b+ and CD61+BMDCs in the 5 mg/kg GEM group also increased slightly, but the change did not reach statistical significance relative to control group. The Gr-1+CD11b+BMDCs count in 0.6/0.03 mg/kg GEM + CDDP-treated B16-tumor-bearing C57BL/6J mice was markedly increased by 23.5% relative to control (p < 0.05) (Fig. 5d). The numbers of CD61+BMDCs increased by 5.3% and 10.9% in the 0.6/0.03 and 4.8/0.24 mg/kg GEM + CDDP groups, respectively, relative to control group (both p < 0.05) (Fig. 5e).
These results indicate that continuous low-dose administration of GEM and GEM + CDDP can significantly mobilize proangiogenic Gr-1+CD11b+ and CD61+BMDCs into the circulating blood in tumor-bearing mice models.
Continuous low-dose administration of GEM and GEM + CDDP promoted the recruitment of BMDCs in tumor tissues
Tumor growth in C57BL/6J mice with GFP bone marrow was observed after 4-weeks of treatment with frequency of once every 2 days with antineoplastic agents. Tumor weights in the 1.25 mg/kg GEM and 0.6/0.03 mg/kg GEM + CDDP groups were increased by 342.5% and 344.9%, respectively, relative to control (both p < 0.01), while they were decreased by 61.8% and 12.9% in the 5 mg/kg GEM and 4.8/0.24 mg/kg GEM + CDDP groups, respectively (Fig. 6a).
The densities of GFP+BMDCs were analyzed using immunohistochemically of tumor tissue sections. Compared with the control group, the numbers of GFP+ cells were increased by 185.8% and 227.8% after treatment with 1.25 mg/kg GEM and 0.6/0.03 mg/kg GEM + CDDP, respectively, compared to control group (both p < 0.01) (Fig. 6b).
These results indicate that the continuous low-dose administration of GEM and GEM + CDDP can promote the recruitment of GFP+ BMDCs in tumor tissues.
Continuous low-dose administration of GEM and GEM + CDDP promoted the expression of proangiogenic proteins in tumor tissues
To confirm the role of low-dose GEM and GEM + CDDP in promoting angiogenesis, we also surveyed the expression changes of some angiogenic proteins in B16 tumor tissues by Immunoblotting. As shown in Fig. 7, the protein expression levels of MMP-9, VE-cadherin, VEGFR1, and VEGFR2 in the 1.25 mg/kg GEM group were increased by 66.6%, 124.3%, 87.6%, and 54.5%, respectively, relative to control (each p < 0.01). In addition, except MMP-9, the protein expression levels of VE-cadherin, VEGFR1, and VEGFR2 in tumor tissues were significantly increased by 65.0%, 30.0%, and 25.0%, respectively, after treatment with 0.6/0.03 mg/kg GEM + CDDP as compared with control (each p < 0.05).
These results suggest that the continuous low-dose administration of GEM and GEM + CDDP can promote the expression of proangiogenic proteins.
ATRA inhibited enhancement of B16 tumor growth and mobilization of BMDCs induced by continuous low-dose administration of GEM
As shown in Fig. 8a, while 1.25 mg/kg GEM treatment resulted in a 75.7% increase in tumor weights relative to control (p < 0.01), co-treatment with 30 mg/kg ATRA led to a 71.2% decrease relative to GEM treatment alone (p < 0.01). Similarly, compared to increased Gr-1+CD11b+ and CD61+BMDCs counts in 1.25 mg/kg GEM group, co-treatment with 30 mg/kg ATRA profoundly inhibited Gr-1+CD11b+ counts 35.8%, and CD61+BMDCs counts by 79.1% (p < 0.01) (Fig. 8b). Meanwhile, as shown in Fig. 8c, the areas of laminin positive vessels in 1.25 mg/kg GEM were significantly enhanced and decreased in 30 mg/kg ATRA in B16 tumor tissues relative to control (both p < 0.01).
These results suggest that low-dose GEM induces mobilization of proangiogenic BMDCs.