Abstract
Background/Aim: The topoisomerase 1 catalytic inhibitor 3EZ, 20Ac-ingenol specifically induces apoptosis through the activation of ATR and the up-regulation of PTEN by enhancing the DNA damage response (DDR) in human B lymphoma (BALL-1) cells. The accumulation of cyclin D1 in cancer is known to be related to chemoresistance to DNA damage agents and nuclei of BALL-1 cells exhibit high levels of cyclin D1. However, 3EZ, 20Ac-ingenol effectively induced apoptosis of BALL-1 cells. Materials and Methods: Cell growth, protein levels, and apoptosis were determined by an MTT assay, immunoblotting and DNA fragmentation assay, respectively. Results: 3EZ, 20Ac-ingenol strongly induced inhibition of cell proliferation and apoptosis in Jeko-1 and Panc-1 cell lines through the activation of tumor suppressor proteins and caspase 3. Conclusion: 3EZ, 20Ac-ingenol-induced apoptosis might occur in cells with cyclin D1 accumulation through enhancing DDR, regardless of the cancer cell type.
- Catalytic topo I inhibitor
- cyclin D1 accumulation
- ATR
- PTEN
- Akt
- GSK-3β
Cyclin D1, encoded by the CCND1 gene located on chromosome 11q13, plays an important role in regulating cell cycle progression (1). This protein promotes the progression of the cell cycle from the G1 to the S phase (2). Therefore, the overexpression of cyclin D1 might promote cellular proliferation, and its up-regulation during oncogenesis might accelerate cell cycle progression in human carcinomas. Overexpression of cyclin D1 has been found in many tumor types, even though the mechanism of cyclin D1 accumulation often differs among tumor types, the existence of a link between cyclin D1 overexpression and oncogenesis has been suggested. Overexpression of cyclin D1 protein in mantle cell lymphoma reportedly occurs as a result of the amplification of the 11q13 region (3). Alternatively, the expression of oncogenes, such as K-ras, is frequently noted during the development of pancreatic cancer (4). The PI3K/Akt/mTOR pathway is a major signaling pathway that mediates the effect of K-ras (5). In addition, Akt-mediated phosphorylation of GSK-3β decreases GSK-3β catalytic activity, inhibiting nuclear export and cytoplasmic degradation of cyclin D1 (6). Cyclin D1 accumulation is reportedly found in 90% of mantle cell lymphoma (7) and 65% of pancreatic cancers (8).
We identified a novel topo I catalytic inhibitor, 3EZ, 20Ac-ingenol, that causes intense phosphorylation of H2AX and induces the DNA damage response (DDR). 3EZ, 20Ac-ingenol has been shown to inhibit cellular proliferation more strongly in chicken (DT 40 cells) and human (BALL-1 cells) B lymphoma than in a myelogenous leukemia cell line, TKG0210, or a T-cell leukemia cell line, TKG0377 (9, 10). Furthermore, we have found that 3EZ, 20Ac-ingenol induces apoptosis during the G2 phase by inhibiting p-Akt and the activation of caspase 3 in DT 40 cells (9), and during the S phase by activating ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) and the up-regulation of Phosphatase and tensin homologue (PTEN) in BALL-1 cells, which exhibit accumulation of cyclin D1 (10, 11). These results suggested that 3EZ, 20Ac-ingenol may act as a catalytic inhibitor that targets B lymphoma cells, cells that accumulate cyclin D1 or cells carrying a mutation that results in the amplification of cyclin D1, as shown previously (3-5, 7, 8), thereby enhancing the DDR and inducing apoptosis.
To examine the specificity of the effect of 3EZ, 20Ac-ingenol on apoptosis, we selected both a mantle cell lymphoma cell line (Jeko-1) and a pancreatic cancer cell line (Panc-1), since both of these cancer cell lines have well-known but different mechanisms of cyclin D1 accumulation. If 3EZ, 20Ac-ingenol selectively induces apoptosis in a manner that depends on the accumulation of cyclin D1, but not on the mechanism of cyclin D1 accumulation, then apoptosis would be observed in both Jeko-1 and Panc-1 cells. If, however, 3EZ, 20Ac-ingenol selectively induces apoptosis in a manner that depends on the mechanism of cyclin D1 accumulation, then apoptosis would be observed in one of the two cell lines. Finally, if 3EZ, 20Ac-ingenol selectively induces apoptosis in a manner that depends on the nature of B-cell lymphoma per se, then apoptosis would only be seen in Jeko-1 cells.
Materials and Methods
Cell lines and cell proliferation. Jeko-1 cells were obtained from the American Type Culture Collections (Rockville, MD, USA). Panc-1 cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The diterpene compound, 3-O-(2’E,4’Z-decadienoyl)-20-O-acetylingenol (3EZ, 20Ac-ingenol) was dissolved in dimethyl sulfoxide. The cancer cells were incubated in RPMI 1640 supplemented with 10% fetal calf serum for 48 h at 37°C. Cell growth was determined by an MTT assay using the Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannheim, Germany) as described previously (9).
Immunoblotting. Jeko-1 cells were cultured for various time periods in the presence of 0.5 μM 3EZ, 20Ac-ingenol, which induced 50% inhibition of cell proliferation after 48 h. Panc-1 cells were also cultured for various time points in the presence of 3 μM 3EZ, 20Ac-ingenol, which induced 50% inhibition of the cell proliferation after 48 h. The cells were washed with PBS and lysed with RIPA buffer (Sigma, MO, USA). Fractionation into a nuclear and cytoplasmic fraction of the cells was performed using Nuclear Cytoplasmic Extraction Reagents (Thermo Scientific, IL, USA). The protein concentrations were determined using the Bradford reagent for protein assays (Bio-Rad Laboratories, CA, USA). A total of 20 μg or 40 μg of the cell lysates were resolved on 8%, 10%, or 15% SDS-polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane. The membranes were incubated with anti-PTEN, anti-p53, anti-p-p53 (Ser15), anti-GSK-3β, anti-p-GSK-3β (Ser9), anti-Akt, anti-p-Akt (Ser473), anti-p21 (Cell Signaling Technology, MA, USA), anti-cyclin D1 (Santa Cruz Biotechnology, TX, USA), anti-ATR (Santa Cruz Biotechnology; Cell Signaling Technology; Abcam, Cambridge, UK), anti-active caspase-3 (R&D systems, MN, USA), anti-γH2AX and anti-actin (Sigma) antibodies followed by detection, using an enhanced chemiluminescence system.
DNA fragmentation assay. To detect cellular apoptosis, a DNA fragmentation assay was used. The Cell Death Detection ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany) was used to detect any cytoplasmic histone-associated-DNA fragmentations. Jeko-1 cells were plated at 5×104 cells/200 μl per tube and treated with 0.5 μM 3EZ, 20Ac-ingenol at 37°C for 12, 24, 48, or 72 h. Panc-1 cells were plated at 5×104 cells/200 μl per tube and treated with 3 μM 3EZ, 20Ac-ingenol at 37°C for 12, 24, 48, or 72 h. After treatment, the cells were lysed and the cell lysates were collected and added to an ELISA plate. The ELISA protocol was performed according to manufacturer's instructions, and the immunocomplex was measured at 405 nm using a microplate reader.
Results
Effects of 3EZ, 20Ac-ingenol or irinotecan on the proliferation of Jeko-1 and Panc-1 cells. The cells were seeded in 96-well plates (2×104 Jeko-1 cells or 3×103 Panc-1 cells per well in 100 μl of medium) and then treated with various concentrations of 3EZ, 20Ac-ingenol (range=0-10 μM). Treatment with 3EZ, 20Ac-ingenol resulted in a concentration dependent decrease in cell viability. Furthermore, for any given concentration, the level of cytotoxicity after 48 h of treatment was higher for the Jeko-1 cells than for the Panc-1 cells. The inhibition of cellular proliferation in the Jeko-1 cell line increased gradually as the concentration of 3EZ, 20Ac-ingenol increased, maximal inhibition of approximately 80% was reached at 1 μM and almost a complete loss of cell viability was observed at 10 μM; the IC50 of the drug for the Jeko-1 cells was about 0.5 μM (Figure 1A). In the Panc-1 cell line, a maximal inhibition of approximately 75% was observed at a concentration of 10 μM; the IC50 for the Panc-1 cells was about 3 μM (Figure 1A).
The effect of the camptothecin analog, irinotecan (CT-11), on the proliferation of the Jeko-1 and Panc-1 cells was examined using the MTT assay (Figure 1B). Treatment with CT-11 inhibited proliferation of Jeko-1 cells and resulted in about 90% reduction in cell viability at 10 μM and to almost 100% reduction of cell viability at 30 μM. However, it caused an almost constant 30% inhibition of the proliferation of Panc-1 cells at concentrations ranging from 10 μM to 50 μM (Figure 1B); the IC50 of the drug for the Jeko-1 cells was about 5 μM, whereas Panc-1 cells were insensitive to irinotecan.
Effects of 3EZ, 20Ac-ingenol treatment on ATR and cyclin A activation, p53 and p-p53 accumulation, phosphorylation of H2AX, and p21 expression. To determine whether DNA damage-sensing kinases and related proteins were activated by 3EZ, 20Ac-ingenol, the protein levels of the primary kinase ATR were examined (Figure 2). In these experiments, a prominent increase in ATR protein levels was observed in whole cell lysates of Panc-1 cells at 12 h after 3EZ, 20Ac-ingenol treatment, but a slight increase was observed in the Jeko-1 cells at 12 h after treatment; the increases in both cell lines continued until 48 h after treatment. p53 protein expression was also elevated in the Panc-1 cells at 12 h after treatment, and the increase continued until 48 h after treatment; the increase in Panc-1 cells was especially pronounced at 24 h after treatment (Figure 2). In Jeko-1 cells, however, an increase in p53 expression was only observed at 48 h after treatment with 3EZ, 20Ac-ingenol. The activation of p53 by its phosphorylation (p-p53) at Ser15 by ATR was analyzed. Although, p-p53 was not detected in whole cell lysates of treated Jeko-1 cells, p-p53 was detected in the Panc-1 cells at 12 h after treatment (Figure 2). We have previously reported that 3EZ, 20Ac-ingenol inhibited cell proliferation during the S phase in BALL-1 cells (10). Cyclin A plays a critical role in the initiation of DNA replication and the subsequent entry of cells into the S phase. We, therefore, examined whether the inhibition of cellular proliferation induced by 3EZ, 20Ac-ingenol through the enhancement of the DDR was accompanied by the regulation of the cell cycle by cyclin A expression. An increase in the cyclin A levels was observed in both the Jeko-1 and the Panc-1 cells at 12 h after treatment with 3EZ, 20Ac-ingenol, and this increase continued until 24 h after treatment; a decrease was then observed at 48 h after treatment (Figure 2).
The phosphorylation of H2AX was examined using anti-γH2AX antibodies in Jeko-1 and Panc-1 cells to determine whether the DDR was induced by 3EZ, 20Ac-ingenol treatment in cells with cyclin D1 accumulation (Figure 2). Time-course analyses in Jeko-1 cells revealed increased phosphorylation of H2AX at 12 h after 3EZ, 20Ac-ingenol treatment (Figure 2), suggesting that 3EZ, 20Ac-ingenol strongly induced the DDR. However, the γH2AX levels decreased at 24 h after treatment. In Panc-1 cells, H2AX phosphorylation was not observed after 3EZ, 20Ac-ingenol treatment.
The expression of p21 in Jeko-1 and Panc-1 cells was examined after induction of DNA damage by 3EZ, 20Ac-ingenol. In Jeko-1 cells, a slight increase in the expression of p21 was observed at 12 h after treatment, with further increases at 24 h and 48 h after treatment (Figure 2). In Panc-1 cells, a pronounced increase in p21 expression was observed at 12 h and 24 h after treatment, although the expression levels were clearly decreased at 48 h after treatment (Figure 2). How these differences in H2AX phosphorylation in Jeko-1 cells and p21 expression in Panc-1 cells were induced during the DDR remains unclear.
Effects of 3EZ, 20 Ac-ingenol treatment on PTEN/Akt signaling and phosphorylation of GSK-3β. Treatment of Jeko-1 and Panc-1 cells with 3EZ, 20Ac-ingenol led to an increase in the PTEN levels in whole-cell lysates at 12 h after treatment elevation persisted until 24 h and then showed a slight decrease at 48 h after treatment (Figure 3A). Next, the effects of 3EZ, 20Ac-ingenol treatment on PI3K/Akt signaling were examined. The levels of phosphorylated Akt (Ser473) in whole-cell lysates of Jeko-1 cells were minimally affected at 12 h after treatment and slightly reduced at 24 h and 48 h after treatment. The expression levels of total Akt protein in Jeko-1 cells also showed little or no change (Figure 3A). Increased levels of p-Akt were observed in whole-cell lysates of Panc-1 cells treated with 3EZ, 20Ac-ingenol until 24 h after treatment, but a slight reduction in p-Akt levels was observed at 48 h after treatment. The expression levels of total Akt protein in Panc-1 cells were also increased at 12 h and 24 h after treatment, but a reduction in total Akt was observed at 48 h after treatment, consistent with slightly constitutive p-Akt inhibition (Figure 3A). After fractionation in nuclear and cytoplasmic fractions, the changes in the nuclear and cytoplasmic p-Akt levels were analyzed. The levels of nuclear p-Akt in Jeko-1 cells also showed a slight decrease at 48 h after treatment; the cytoplasmic p-Akt levels in Jeko-1 cells was clearly reduced at 12 h after treatment, and the reduced cytoplasmic p-Akt levels persisted at 24 h and 48 h after treatment (Figure 3A). Transient increases in the p-Akt levels were observed in the nuclear and cytoplasmic fractions of Panc-1 cells at 12 h and 24 h after treatment, followed by a reduction in the levels at 48 h after treatment (Figure 3A). The effect of treatment on the phosphorylation of GSK-3β, a downstream target of Akt, was examined. Our results revealed that 3EZ, 20Ac-ingenol treatment reduced in decreased phosphorylation of GSK-3β (Ser9) in Jeko-1 cells at 24 h after 3EZ, 20Ac-ingenol treatment, and the p-GSK-3β levels continued to decrease until 48 h after treatment (Figure 3B). Panc-1 cells also exhibited a decrease in the p-GSK-3β levels at 24 h after treatment, and a further decrease was observed at 48 h after treatment (Figure 3B). In contrast, an increase in GSK-3β levels in Jeko-1 and Panc-1 cells treated with 3EZ, 20Ac-ingenol was observed at 12 h after treatment and continued thereafter (Figure 3B).
Effects of 3EZ, 20Ac-ingenol treatment on the accumulation of cyclin D1. Although, the mechanism of cyclin D1 accumulation in BALL-1 cells remains unclear, we found that cyclin D1 accumulation was reduced with 3EZ, 20Ac-ingenol treatment (10). High levels of cyclin D1 accumulation were also observed in untreated whole lysates of Jeko-1 cells, while lower levels of accumulation were detected in untreated Panc-1 cells (Figure 4). A similar gradual decrease in the accumulation of cyclin D1 was observed after treatment in Jeko-1 and Panc-1 cells. The control Jeko-1 cells showed relatively high levels of nuclear cyclin D1; in the treated cells, the levels were gradually decreased from 12 h to 48 h after treatment with 3EZ, 20Ac-ingenol (Figure 4). Although the cytoplasmic cyclin D1 levels were also high in the control Jeko-1 cells, a decrease was observed after 3EZ, 20Ac-ingenol treatment and cytoplasmic cyclin D1 level was no longer detectable at 48 h after treatment (Figure 4). Although the levels of nuclear cyclin D1 level were lower in control Panc-1 cells, a transient increase in the nuclear cyclin D1 levels was observed at 12 h and 24 h after treatment with 3EZ, 20Ac-ingenol and then decreased to a lower level at 48 h after treatment (Figure 4). Cytoplasmic cyclin D1 was not detectable in control Panc-1 cells, but a transient increase was observed at 12 h and 24 h after 3EZ, 20Ac-ingenol treatment and then similar to nuclear cyclin D1 decreased and became undetectable at 48 h after treatment (Figure 4). These results in Jeko-1 and Panc-1 cells might reflect the nuclear export and cytoplasmic degradation of cyclin D1 by 3EZ, 20Ac-ingenol.
Effects of 3EZ, 20Ac-ingenol treatment on caspase-3 activation and apoptosis induction. To determine whether the inhibition of proliferation of Jeko-1 and Panc-1 cells by 3EZ, 20Ac-ingenol was associated with the induction of caspase activation and apoptosis, a western blot analysis was performed using antibodies against the cleaved forms of caspase-3. Caspase-3 activation was first observed at 48 h after treatment in Jeko-1 cells, and at 12 h after treatment in Panc-1 cells; further activation was observed in Panc-1 cells at 24 h and at 48 h (Figure 5). p53 accumulation in Jeko-1 cells occurred relatively late (Figure 2), suggesting that caspase 3 activation might also occur late.
To detect cellular apoptosis we carried out a DNA fragmentation assay. An increased number of DNA fragments was observed in Jeko-1 cells treated with 0.5 μM 3EZ, 20Ac-ingenol beginning at 48 h after treatment and continuing until 72 h after treatment (Figure 6A). In the presence of 3 μM 3EZ, 20Ac-ingenol, an increase in the number of DNA fragments in Panc-1 cells was observed at 24 h after treatment, and further increases were observed at 48 h and 72 h (Figure 6B). Higher levels of DNA fragmentation were observed in Panc-1 cells, compared with Jeko-1 cells. This difference could be related to cell size, since Jeko-1 cells are relatively small and Panc-1 cells are relatively large.
Discussion
The catalytic topo I inhibitor 3EZ, 20Ac-ingenol induced apoptosis by enhancing the DDR in both a B-cell lymphoma cell line (Jeko-1), as characterized by H2AX phosphorylation, and a pancreatic cancer cell line (Panc-1), as characterized by the up-regulation of p21 expression.
The major regulators of the DDR signaling pathway responding to DNA damage induced by topo 1 poison inhibitors are ATM and ATR, which drive the cytotoxic effects of topo poisons by activating H2AX phosphorylation, Chk phosphorylation and p53 accumulation (12-14). In particular, ATR is activated during the S-phase of the cell cycle to regulate replication initiation and the repair of damaged replication forks (15-17). Although the induction of DDR signaling by topo poison inhibitors is characterized by H2AX phosphorylation and the activation of the ATR-p53 pathway, the same DDR was induced by the topo catalytic inhibitor 3EZ, 20Ac-ingenol in both Jeko-1 and Panc-1 cells. p53 accumulation and its activation by phosphorylation via ATM and Chk2 as a result of DNA damage (18, 19) contributes to the release of cytochrome c from the mitochondria via both transcription-dependent and transcription-independent mechanisms, resulting in the activation of caspase 3 (20, 21). In addition to the activation of ATR signaling, 3EZ, 20Ac-ingenol also caused the down-regulation of p-Akt and p-GSK-3β through the up-regulation of PTEN, thereby inducing apoptosis in both Jeko-1 and Panc-1 cells. Many topo catalytic inhibitors that induce a decatenation checkpoint causing G2 phase arrest are less toxic and, therefore, cannot induce apoptosis. Betulinic acid does not stabilize the enzyme-DNA cleavable complex directly but indirectly lead the enzyme-DNA cleavable complex through the generation of reactive oxygen species. Nevertheless, it does not affect cell death (22). Another topo I inhibitor, CY13II, inhibits topo-1 mediated DNA cleavage in a manner similar to that of 3EZ, 20Ac-ingenol, acting as a catalytic inhibitor to induce G2/M phase arrest and cell growth (23). Since the activation of Akt (p-Akt) in response to DNA damage occurs through the same signal transduction pathways as the activation of the PI3 kinase-like kinases, ATM and ATR (24, 25), Akt might also be activated in Jeko-1 and Panc-1 cells (Figure 3), and activation might be reduced by up-regulated PTEN at 48 h after treatment, ultimately resulting in transient activation of Akt. Catalytic topo inhibitors that up-regulate PTEN in addition to clearly activating the ATR pathway have not been previously reported, and the activation mechanism for the decatenation checkpoint cascade remains to be clarified. The induction of apoptosis through a decrease in cyclin D1 in BALL-1 cells in response to treatment with catalytic inhibitors would be impossible if 3EZ, 20Ac-ingenol did not enhance the DDR in these particular cells (10, 11). We searched for the cause of the enhancement of the DDR in BALL-1, Jeko-1 and Panc-1 cells. We observed that 3EZ, 20Ac-ingenol induced H2AX phosphorylation, which in turn increased p53 accumulation and caspase 3 in BALL-1 (10) and Jeko-1 cells, whereas it increased p21 expression, which in turn increased p-p53 accumulation in Panc-1 cells (Figure 2) and although mutations of p53 are present in Jeko-1 and Panc-1 cells (26-28), increased the expression of downstream proteins and activated caspase 3. Reportedly, cyclin D1 overexpression enhances the DDR, as characterized by the induction of H2AX phosphorylation by topo inhibitors and the increase in p21 expression (29), and cells that retain cyclin D1 in their nuclei are sensitive to topo inhibitor (30). We became convinced that 3EZ, 20Ac-ingenol specifically inhibits cell proliferation and induces apoptosis by enhancing the DDR in cancer cells with accumulated cyclin D1 based on results showing that these effects were observed in three different cancer cells of different origins including BALL-1 (10), in which cyclin D1 accumulation and an enhanced DDR were observed. Our results suggested that 3EZ, 20Ac-ingenol may promote ATR activation in addition to the down-regulation of p-Akt and p-GSK-3β via the up-regulation of PTEN by enhancing the DDR in cells with cyclin D1 accumulation, thereby inducing apoptosis in these cells through the degradation of cyclin D1.
Cyclin D1 regulates normal progression of the cell cycle from the G1 to the S phase (1, 2); after progression to S phase, the removal of cyclin D1 from the nucleus is essential for regulating cell division (6). Cyclin A-associated Cdk2 play a critical role in the initiation of DNA replication and subsequent S phase entry (31). We assessed the effect of 3EZ, 20Ac-ingenol on the expression of cyclin A and cyclin D1 proteins during cell cycle progression to S phase. Progression to S phase following 3EZ, 20Ac-ingenol treatment was observed, and increased levels of cyclin A were observed in both the Jeko-1 and Panc-1 cell lines (Figure 2); although the levels of nuclear cyclin D1 should have been significantly reduced, the nuclei of these cells instead retained cyclin D1 (Figure 4). We next considered whether the retention of cyclin D1 compromises the response and the transient activation of nuclear cyclin D1 in Panc-1 cells (Figure 4). These activities might promote chromosomal instability in the nuclei of Jeko-1 and Panc-1 cells, terminating the progression of the cell cycle at the S phase (15). PTEN is known to play a role in activating DNA repair to maintain chromosomal stability (32, 33). The DNA repair reaction to maintain chromosomal stability might be strongly induced in both cell types after 3EZ, 20Ac-ingenol treatment, together with the activation of ATR and the upregulation of PTEN. Numerous reports have suggested that p21 may act as an anti-apoptotic or pro-apoptotic effector in the response to DNA damage (34). Human cancer cell lines treated with DNA damaging agents undergo cell cycle arrest mediated by p21, followed by apoptosis after the caspase-3 mediated cleavage of p21 (35). p21 expression induced by various drugs is reportedly associated with cytochrome c release (36) or the upregulation of the active subunit of caspase-3 (37, 38), and activated caspase-3 cleaves p21 (39), followed by apoptosis. We also observed accumulation of p53 (Figure 2) and p21 (Figure 2) in Panc-1 cells at 12 h after treatment with 3EZ, 20Ac-ingenol and association with the activation of caspase-3 at 12 h after treatment (Figure 5). The levels of p21 were decreased in the Panc-1 cells between 24 h and 48 h after treatment (Figure 2), and apoptosis of the Panc-1 cells was induced beginning at 24 h after treatment (Figure 6B).
In comparison to other topo poisons, treatment with 10 μM 3EZ, 20Ac-ingenol led to a complete loss of cell viability of Jeko-1 cells. While the inhibitory effect on cell proliferation was less pronounced in Panc-1 cells than in Jeko-1 cells, a substantial decrease in the viable cell population was also observed in Panc-1 cells. Although pancreatic cancers and mantle cell lymphoma with cyclin D1 accumulation are insensitive or resistant to DNA damage drugs (40-44), the knockdown of cyclin D1 has been shown to restore sensitivity to these agents (42-44). Jeko-1 and Panc-1 cells, which exhibit cyclin D1 accumulation, are naturally resistant to chemotherapy, but treatment with 3EZ, 20Ac-ingenol restored drug sensitivity irrespective of the mechanism of cyclin D1 accumulation by decreasing the cellular accumulation of cyclin D1, thereby inhibiting cell proliferation (Figures 1A and B) and inducing apoptosis (Figures 5 and 6). p53 is the most frequently mutated gene in pancreatic cancer, and its mutations result in loss of wide-typep53 function, contributing to malignant progression (26, 28). Recently, mutant p53 has become an attractive target for cancer therapy (45). Reportedly, it also induces apoptosis through activation of mutant p53 (46-48). 3EZ, 20Ac-ingenol also significantly increased the expression of mutant p53 protein and the p53-downstream target gene p21 and the activation of caspase 3 in Panc-1 cells. Future study of this pathway could provide important information for the development of chemotherapeutic agents for cancers exhibiting dysregulated cyclin D1 expression.
Acknowledgements
This investigation was supported in part by a grant from Nihon University to S. Miyata.
Footnotes
Authors' Contributions
All Authors performed the experiments and collaborated in the writing of this paper.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received September 4, 2020.
- Revision received September 16, 2020.
- Accepted September 18, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved