A synthetic podophyllotoxin derivative exerts anti-cancer effects by inducing mitotic arrest and pro-apoptotic ER stress in lung cancer preclinical models

Abstract

Some potent chemotherapy drugs including tubulin-binding agents had been developed from nature plants, such as podophyllotoxin and paclitaxel. However, poor cytotoxic selectivity, serious side-effects, and limited effectiveness are still the major concerns in their therapeutic application. We developed a fully synthetic podophyllotoxin derivative named Ching001 and investigated its anti-tumor growth effects and mechanisms in lung cancer preclinical models. Ching001 showed a selective cytotoxicity to different lung cancer cell lines but not to normal lung cells. Ching001 inhibited the polymerization of microtubule resulting in mitotic arrest as evident by the accumulation of mitosis-related proteins, survivin and aurora B, thereby leading to DNA damage and apoptosis. Ching001 also activated pro-apoptotic ER stress signaling pathway. Intraperitoneal injection of 2 mg/kg Ching001 significantly inhibited the tumor growth of A549 xenograft, while injection of 0.2 mg/kg Ching001 decreased the lung colonization ability of A549 cells in experimental metastasis assay. These anti-tumor growth and lung colonization inhibition effects were stronger than those of paclitaxel treatment at the same dosage. The xenograft tumor tissue stains further confirmed that Ching001 induced mitosis arrest and tumor apoptosis. In addition, the hematology and biochemistry tests of blood samples as well as tissue examinations indicated that Ching001 treatment did not show apparent organ toxicities in tested animals. We provided preclinical evidence that novel synthetic microtubule inhibitor Ching001, which can trigger DNA damage and apoptosis by inducing mitotic arrest and ER stress, is a potential anti-cancer compound for further drug development.

Figure 1. Ching001 selectively inhibits proliferation and delays M-phase progression.

(A) The structure of Ching001 compound. (B) Cytotoxicity assay of normal lung MRC5 and various lung cancer cells was monitored by trypan blue staining. Cells were treated with different concentration of Ching001 for 48 h. Data represent mean ± SEM from three independent experiments. Asterisks indicate significant differences between the normal MRC5 cells and cancer cells. *** P<0.001. (C) Flow cytometry analysis of cell cycle distribution of lung cancer cell lines treated with varying doses of Ching001 and for different durations. (D) Immunofluorescence for α-tubulin (green) and DAPI nuclear staining (blue) after 1 µM Ching001 treatment for 2 to 8 h in S-phase synchronized lung cancer cell lines. DMSO was used as solvent control. Scale bars: 10 µm.

Figure 2. Ching001 induces M-phase arrest and DNA damage.

(A) Western blot analyses of mitosis-related proteins after 1 µM Ching001 treatment for indicated times in lung cancer cell lines. (B) Dysregulation of M-phase cell cycle induced by Ching001. Lung cancer cell lines were analyzed with survivin (green), aurora B (red), and DAPI (blue) after 1 µM Ching001 treatment for 24 h. DMSO was used as solvent control. Scale bars: 30 µm.

Figure 3. Ching001-induced mitotic arrest leads to DNA damage and apoptosis of lung cancer cells.

(A) Western blot analyses for DNA damage marker γ–H2AX after 1 µM Ching001 treatment at indicated times. (B) Immunofluorescence for early-apoptotic marker PS translocation after Ching001 treatment for 24 h. Scale bars: 30 µm. (C) The apoptotic-specific DNA fragmentation was detected by DNA ladder analyses after Ching001 treatment for 48 h.

Figure 4. Ching001 induces ER stress mediated apoptosis.

(A) The caspase-4 activity increased time-dependently after 1 µM Ching001 treatment in both A549 and H1299 lung cancer cell lines at indicated times. **: P<0.01, ***: P<0.001. (B) Western blot analyses of ER stress related signaling proteins (blots above the dot line) and apoptosis related signaling proteins (blots below the dot line) after Ching001 treatment at the indicated times.

Figure 5. Ching001 inhibits A549 xenograft growth via M-phase arrest and apoptosis.

(A) ICR-nude mice bearing the established A549 tumors (∼50 mm³) were treated with Ching001 (0.4 mg/kg or 2 mg/kg) via intraperitoneal injection on day1, day3, day5, day7, and day9 (as indicated by arrow heads). A known microtubule inhibitor, paclitaxel (4 mg/kg), was used for comparison. The tumor volumes (left) and body weight (right) were measured on every other day till day35. Points, mean; bars, ±SEM. *: P<0.05, **: P<0.01, ***: P<0.001. (B) The activated caspase-3 IHC staining of the tumor tissue of ICR-nude mice taken from solvent control group and Ching001 treatment (2 mg/kg) group. Original magnification×200. (C) The tissue immunofluorescence of survivin (green), aurora B (red), and DAPI (blue) of tumor xenograft from solvent control mice and Ching001 treated mice (2 mg/kg). The arrows indicated the mitotic cells in solvent control tumor. The enlarged figure represents aberrant chromosomes after Ching001 treatment. Scale bars: 30 µm.

Figure 6. Ching001 inhibits colonization of A549 lung cancer cell in animal models without significant side effects.

(A) A549 cells (1×10⁶) were tail vein injected into the BALB/c mice. The mice received 0.2 mg/kg Ching001 or 0.2 mg/kg Paclitaxel intraperitoneally every-other day for five weeks as indicated by arrow heads in lower right panel. Cells pretreated with 1 µM Ching001 before tail vein injection were also performed. DMSO was used as solvent control. The H&E-staining of the lung tissue (left), quantification of the tumor nodules in the lungs (upper right), and body weight (lower right) of A549 injected mice are shown. The red arrows indicated the sites of tumor nodules in the lung tissue. Original magnification×100. *: P<0.05. (B) The hematology and biochemistry tests of blood from tested mice. In the hematology tests, WBC, RBC, and platelet were tested. In biochemistry tests, GOT, GPT, albumin, and total-bilirubin are used as indicators of liver function; BUN and creatinin as indicators of renal function. The data indicated that Ching001 treatment caused no apparent change on liver and kidney functions compared to DMSO-treated animals.

Figure 7. Summary of the possible anti-tumor mechanisms of Ching001.

Rapid cell cycle progression is one of the features of the proliferated cancer cell. Inhibition of microtubule polymerization by Ching001 arrests M-phase cell cycle progression and leads to DNA damage. In addition, Ching001 treatment triggered activation of ER stress signaling pathway through the activation of PERK and caspase-4 signaling cascade. Prolonged M-phase arrest and activated ER stress signaling subsequently induce apoptosis in the cancer cell treated with Ching001.