Figure 1.
Distinct effects of FL118 on DNA topoisomerase 1 (Top1) activity and cancer cell growth.
a. Structure of the camptothecin analogs FL118 (MW: 392) and of irinotecan (MW: 587). b. FL118 inhibits DNA Top1 to a similar extent as the active form of irinotecan SN-38. A representative result tested at 1 µM (the highest concentration that can be reached by irinotecan in vivo) is shown. Experiments were performed as described in Methods. c and d. FL118 effectively inhibits cancer cell growth at sub-nM levels. Cancer cells at 50–70% confluence were treated with vehicle (DMSO) or FL118 at different concentrations as shown in c and d. Cell growth/viability were determined by MTT assay 72 hours after treatment. Each bar is the mean ± SD (standard deviation) derived from independent assays (N = 4) performed in at least triplicate. Representative statistical p-values are indicated. e and f. Comparison of the effects of FL118 and the irinotecan active form SN-38 on survivin expression in two cancer cell types with different p53 status. Subconfluent cancer cells with distinct p53 status were treated with or with SN-38 or FL118 for 24 hours as shown. Cells were then lysed and analyzed using Western blots with corresponding antibodies as shown (e, f). Actin was used as internal controls.
Figure 2.
Cancer cells with high survivin expression are significantly more sensitive to FL118 treatment than non-cancerous cells, which have low or no survivin regardless of p53 status.
Cells at about 50% confluence were treated with or without FL118 in a series of concentrations as shown (a, c) for 72 hours. Cell growth/viability were then determined using MTT assay and plotted as percentage cell viability curves. Alternatively, cells at 80–90% confluence were lysed and analyzed using Western blots for survivin expression (b, d). a. Cell viability curve for two cancer cell lines (SW620, HCT-8) versus two non-cancerous cell lines (AHDF, HGF) after FL118 treatment for 72 hours at the different concentrations as shown. b. Comparison of the expression of survivin among two cancer cell lines (SW620, HCT-8) versus two non-cancerous cell lines (AHDF, HGF). Actin was used as an internal control. c. Cell viability curve for additional two cancer cell lines (2008, FaDu) versus one non-cancerous cell line (AHDF) as a control after FL118 treatment for 72 hours at different concentrations as shown. d. Comparison of the expression of survivin in additional two cancer cell lines (2008, FaDu) versus one non-cancerous cell lines (AHDF) as a control. Actin was used as an internal control. The data on each data point shown in a and c are the mean ± SD derived from at least three independent assays in triplicate.
Figure 3.
Treatment of cancer cells with FL118 significantly decreases BrdU positive cells (proliferative cells) and induces apoptotic markers.
HCT-8 cells at 50% confluence were treated with or without FL118 (10 nM) for 24 hours and were labeled with BrdU in the last 40 min of FL118 treatment. Cells were then stained with 7AAD, followed by flow cytometry analysis. a. A representative example is shown. b. Statistical results derived from three independent flow cytometry analyses. c. FL118 induces caspase-3 activation and PARP cleavage, hallmarks of apoptosis. Sub-confluent HCT-8 colon cancer cells were treated with or without FL118 at the concentration and time points shown. Cells were then lysed and analyzed by western blot. Actin expression was used as an internal control. Quantification as %Control: 100, 101, 93, 21, 4 for survivin; 1, 1, 2, 8, 13 for activated caspase 3; and 1, 2, 3, 31, 49 for cleaved PARP.
Figure 4.
FL118 selectively inhibits human survivin promoter activity and endogenous survivin expression.
a and b. FL118 inhibits survivin promoter activity at concentrations as low as 0.1–1 nM. Cancer cells that stably express a full-length survivin promoter (4080 bp)-driven luciferase construct were treated with FL118 for 24 hours at different concentrations as shown, followed by luciferase activity assay. c, d and e. FL118 does not inhibit promoter activity for the genes of p21, DHFR, HTR or TK at 10 nM levels. Sixteen hours after transfection, cells were treated with FL118 (10 nM) for 24 hours, followed by luciferase activity assay. Each bar (a–e) is the mean ± SD derived from independent testing (N = 3) of at least triplicates. f and g. FL118 inhibits endogenous survivin expression but does not inhibit expression of endogenous p21 and DHFR proteins. Representative results are shown. Subconfluent EKVX (f) and HCT-8 (g) cells were treated with and without FL118 for 24 hours, then cell lysates were analyzed by Western blot. Actin expression was used as an internal control. Quantification: f. %Control: 100, 32, 15 for survivin; 100, 255, 165 for p21; and 100, 91, 97 for DHFR. g. %Control: 100, 48, 32 for survivin; 100, 95, 99 for p21; and 100, 101, 112 for DHFR.
Figure 5.
FL118 selectively and differentially modulates the expression of IAP and Bcl-2 family proteins.
Subconfluent cells were treated with and without FL118 at the concentration and time points as shown (a–c). Cells were then lysed and analyzed by western blot. Representative results are shown. a. A time course for FL118-mediated inhibition of survivin expression in 2008 ovarian cancer cells. b. Differential modulation of the expression of antiapoptotic and proapoptotic proteins by FL118 in PC-3 prostate cancer cells. Quantification as %Control: 100, 7, 5 for survivin; 100, 9, 12 for Mcl-1; 100, 88, 77 for Bcl-2; 100, 54, 49 for Bcl-XL; 1, 100, 43 for Bax; 1, 18, 74 for Bim. c. Downregulation of survivin, XIAP, cIAP2 and Mcl-1 by FL118 in HCT-8 colon cancer cells. The “*” in the survivin panel indicates the potential expression of the proapoptotic protein survivin-2B after FL118 treatment. Quantification as %Control: 100, 11, 7 for survivin (of note, the asterisk-marked band excluded); 100, 9, 8 for XIAP; 100, 22, 12 for cIAP2; and 100, 13, 6 for Mcl-1. d. Genetic silencing of survivin does not affect the expression of other FL118 downstream targets. HCT-8 colon cancer cells were infected with lentiviral survivin-shRNA for 48 hours, followed by western blot analysis of the expression of FL118 downstream targets. Quantification as %Control: 100, 13 for survivin; 100, 96 for XIAP; 100, 98 for cIAP2; and 100, 136 for Mcl-1. Of note, the survivin level without mock lentiviral infection is similar to the mock control lentiviral infected cells (not shown). Actin expression shown in a–d is internal controls. e. FL118 inhibits Mcl-1 promoter-driven luciferase activity. Subconfluent HCT-8 colon cancer cells were transiently transfected with pGL2-Mcl-1. The transfected cells were treated with or without FL118 for 24 hours 16 hours post transfection. Each bar is the mean ± SD derived from three independent assays.
Figure 6.
Effect of FL118 on Bim and Bax mRNA and HDAC activity.
a and b. Effect of FL118 on Bim and Bax mRNA. Subconfluent HCT-8 cells were treated with or without FL118 for 24 hours, followed by isolation of total RNA for real-time qPCR using either Bim primers (a) or Bax primers (b) following the procedures described in the Methods. Data is plotted as a histogram. Each bar is the mean ± SD derived from three independent assays in triplicate. c. Effect of FL118 on HDAC activity. Nuclear extracts were isolated from subconfluent SW620 cells. The HDAC activity inhibition experiment was performed following the protocol in the Method section. The resultant data were plotted in a histogram; each bar is the mean ± SD derived from three independent assays.
Figure 7.
The FL118 downstream target survivin play a role in FL118-mediated inhibition of cancer cell growth and apoptosis.
a Subconfluent HCT-8 colon cancer cells were infected with a lentiviral delivery system containing mock shRNA (shRNA-EGFP) or survivin shRNA. After puromycin selection at 2 µg/ml, the puromycin-selected infectants were treated with or without FL118 for 72 hours. Cells were then analyzed using the MTT assay for cell viability. Each bar is the mean ± SD derived from three independent assays. Of note, downregulation of survivin by the lentiviral survivin shRNA was confirmed by western blots (Fig. 5d). b–d. Subconfluent HCT-8 colon cancer cells were infected with lentiviral survivin shRNA particles or control lentiviral particles as above. After up to 7 days selection with puromycin (2 µg/ml), the mixed infectants were treated with or without FL118 (10 nM) for 36 hours. Cells were stained with Annexin V/PI, followed by flow cytometry. b. A representative flow cytometry result gated with PI (Y axis) and Annexin V (Alexa Fluor 647, X axis). c and d. Quantitative data from b for R4 (c) and R2+R4 (d) from three independent measures in parallel. Of note, R1 is Annexin V negative/PI positive cells; R2 is both Annexin V and PI positive cells (later apoptotic cells); R3 is both Annexin V and PI negative cells; and R4 is Annexin V positive/PI negative cells (early apoptotic cells).
Figure 8.
FL118 downstream targets (Mcl-1, XIAP, cIAP2) play a role in FL118-mediated apoptotic marker production and apoptosis.
a Subconfluent HCT-8 colon cancer cells were infected with a lentiviral delivery system containing mock shRNA (shRNA-EGFP) or Mcl-1 shRNA. After puromycin selection, the puromycin-selected infectants were treated with or without FL118 for 36 hours. Cells were then analyzed using western blot for PARP cleavage. Quantification as %Control: 100, 8, 57, 6 for Mcl-1; 1, 2, 1, 18 for cleaved PARP. b and c. Subconfluent HCT-8 colon cancer cells were transfected with or without mammalian expression vector for XIAP (b) cIAP2 (c) overnight, and then treated with or without FL118 (10 nM) for 36 hours. Cells were then lysed and analyzed via western blots to determine caspase-3 activation or PARP cleavage. Quantification as %Control: b. 100, 247, 100, 244 for XIAP; 1, 2, 16, 7 for cleaved PARP. c. 100, 1470, 100, 1645 for cIAP2; 1, 1, 21, 15 for activated caspase 3; and 1, 1, 3, 1 for cleaved PARP. Actin expression was used as internal controls (a–c). d–f. Effect of overexpression of XIAP on FL118-induced Annexin V staining (apoptosis). Subconfluent HCT-8 colon cancer cells were transfected with XIAP expression vectors for 16 hours. The transfected cells were then treated with or without FL118 (10 nM) for 36 hours. Cells were stained with Annexin V/PI, followed by flow cytometry. d. A representative flow cytometry result gated with PI (Y axis) and Annexin V (Alexa Fluor 488, X axis). e and f. Quantitative data from d for R4 (e) and R2+R4 (f) from three independent measures in parallel. Of note, same as in Figure 7, R1 is Annexin V negative/PI positive cells; R2 is both Annexin V and PI positive cells (later apoptotic cells); R3 is both Annexin V and PI negative cells; and R4 is Annexin V positive/PI negative cells (early apoptotic cells).
Table 1.
The maximum tolerated dose (MTD) of FL118 at the schedule of weekly × 4.
Figure 9.
A single injection of FL118 shows superior antitumor activity in comparison with other clinically used antitumor drugs.
a. Results from the human head and neck FaDu tumor xenograft mouse model. b. Results from the human colon tumor HCT-8 xenograft mouse model. Treatment was initiated 7 days after subcutaneous tumor implantation, at which time the tumor volume was about 200–250 mm3 (designated Day 0). Drugs were intraperitoneally (i.p.) administrated on Day 0 (indicated with arrow) at their MTD with a single dose schedule. The dose for FL118 is its MTD for the weekly × 4 schedule (Table 1). The tumor growth curves in response to each compound are shown as the mean tumor size ± SE from five individual mice at each time point.
Figure 10.
Comparison of antitumor efficacy and toxicity (body weight loss) of FL118 with irinotecan in a SCID mouse model of human primary head and neck tumor (17073)-established xenograft.
The tumor model set up was the same as in Figure 9. a. The mean tumor growth curves ± SE derived from five individual mice in response to treatment with vehicle (control), FL118 or irinotecan. b. The mean mouse body weight change ± SE derived from five individual mice in response to treatment with vehicle (control), FL118 or irinotecan. c, d and e. Tumor growth curves from individual tumor xenografts in response to treatment with vehicle (c), irinotecan (d) or FL118 (e). The treatment schedule was weekly × 4 as indicated by arrows. The dose used for irinotecan and FL118 was their corresponding MTD for the weekly × 4 schedule.
Table 2.
Antitumor activity of FL118 versus irinotecan in nude mice bearing human colon and head-&-neck tumor xenografts.
Figure 11.
FL118 effectively eliminates large tumors.
Individual athymic nude mice were xenografted with both human head and neck FaDu tumor on the left side (a) and human colon SW620 tumor on the right side (b). After the tumor grew to their maximal sizes allowed by IACUC (1500–2000 mm3), mice were treated with FL118 at the dose of 1.5 mg/kg weekly × 4 schedule as indicated with arrows. Each curve represents an individual mouse during and after treatment. Of note, one mouse with both FaDu (on left side, a) and SW620 (on right side, b) tumors died on Day 21 for an unknown reason. This could be the result of Tumor Lysis Syndrome (TLS), which can be caused by treatment-induced massive tumor necrosis (http://emedicine.medscape.com/article/989050-overview).