Open Access

Tan IIA inhibits H1299 cell viability through the MDM4‑IAP3 signaling pathway

  • Authors:
    • Yukun Zu
    • Jianning Wang
    • Wei Ping
    • Wei Sun
  • View Affiliations

  • Published online on: November 24, 2017     https://doi.org/10.3892/mmr.2017.8152
  • Pages: 2384-2392
  • Copyright: © Zu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Tanshinone IIA (Tan IIA), as a bioactive compound extracted from the dried roots of Salvia miltiorrhiza (also known as Danshen), is known to inhibit cancer cell proliferation and induce apoptosis. However, the mechanisms underlying the function of Tan IIA in cancer cell apoptosis remain to be elucidated The aim of the present study was to identify the molecular mechanisms underlying the anti‑cancer effects of Tan IIA in p53‑deficient H1299 cells. Tan IIA was demonstrated to suppress murine double minute 4 (MDM4) expression in a time‑ and dose‑dependent manner through the inhibition of MDM4 mRNA synthesis. Tan IIA‑induced downregulation of MDM4 resulted in an increase of P73α and a decrease of inhibitor of apoptosis 3 (IAP3). However, P73α was not activated as two P73α target genes, BCL2 binding component 3 and phorbol‑12‑myristate‑13‑acetate‑induced protein 1, were not significantly induced. Tan IIA‑induced inhibition of IAP3 expression may be involved in Tan IIA‑induced apoptosis and inhibition of H1299 cell viability. Notably, a combination of Tan IIA and doxorubicin (DOX) exposure resulted in further MDM4 overexpression in H1299 cells, indicating that Tan IIA sensitized p53‑deficient and MDM4‑overexpressing H1299 cells to DOX‑induced apoptosis.

Introduction

Salvia miltiorrhiza (SM), also known as Danshen, is a member of the Labiatae family. It is a nontoxic tonic herb used for improving microcirculation in traditional Chinese medicine, and has been used in Asian countries to treat cardiovascular diseases, including myocardial infarction, angina pectoris and atherosclerosis, due to its multiple therapeutic effects (1,2). Tanshinone IIA (Tan IIA) is a bioactive compound extract from the dried roots of SM. Tan IIA has been documented to possess antitumor activity in multiple types of human cancer cell (37). The antitumor activity of Tan IIA is primarily due to the inhibition of proliferation and the induction of apoptosis (3,4). The induction of endoplasmic reticulum stress has also been noted (5). Tan IIA also decreases human cancer cell invasion and metastasis (6). Thus, Tan IIA may be a potential anti-cancer agent. A previous study reported that Tan IIA inhibited the proliferation of non-small cell lung cancer A549 cells, potentially by decreasing the mitochondrial membrane potential and causing apoptosis via to the induction of a higher ratio of BCL2 associated X, apoptosis regulator/B-cell lymphoma 2 (7). However, the mechanisms underlying the anti-cancer activity of Tan IIA remain to be further elucidated.

The p53 tumor suppressor gene is involved in the response to genotoxic stress exposure, including cell cycle arrest, DNA repair, senescence or apoptosis (8). A previous study has demonstrated that >50% of human cancers contain p53 mutations (9). P73 is a p53 family member that generates two groups of isoforms, either containing a complete transactivation domain (TAp73, also named P73α) or exhibiting a truncated TA domain (10). Inhibition of cell proliferation or induction of cell apoptosis is induced by P73α in response to treatment with anti-cancer agents, including doxorubicin (DOX) (11). However, in contrast to p53, failure of tumor-formation in p73 knockout mice and observations of p73 overexpression in tumor cells, do not support p73 as a classical tumor suppressor (12,13). Thus, the functions of p73 remain to be further elucidated.

Murine double minute (MDM)2, as an antagonist of p53/p73, is involved in downregulating p53/p73 activity (14). MDM2 inhibits p53 activity by binding to the N-terminal domain of p53 and blocking p53-dependent transcriptional activity, or by ubiquitination of p53 and targeting it for proteasomal degradation (15). As with p53-MDM2 interaction, binding of the P73 and MDM2 also results in the suppression of p73-transcriptional activity (16). However, MDM2 does not ubiquitinate p73 (17). MDM2 overexpression has been observed in 7% of all human cancers, with higher frequencies in soft tissue tumors, osteosarcomas and esophageal carcinomas (18).

MDM4, also known as MDMX, is a homolog of MDM2. Similar to MDM2, MDM4 also binds to and inhibits p53-and p73-dependent transactivation (14). However, unlike MDM2, MDM4 does not demonstrate appreciable ubiquitin ligase activity (14). MDM4 overexpression has been reported in ~17% of mantle cell lymphomas, breast cancers, uterine cancers, testicular cancers, stomach/small intestinal cancers, colorectal cancers, lung cancers and malignant melanomas (19). However, the effects of MDM4 on tumor properties, as well as its function during tumorigenesis, remain unknown.

Although the mechanisms underlying MDM2 and MDM4 overexpression in human cancers are not known, overexpression of the two proteins has been demonstrated to be associated with the promotion of cancer and poor treatment outcome (20,21). In the present study, the influence of Tan IIA exposure on MDM2 and MDM4 expression and the inhibition of cell proliferation was investigated in a p53-deficient cell model using the H1299 cell line. The data demonstrated that Tan IIA downregulated expression of MDM4, but not MDM2, and inhibited the viability of H1299 cells. Herein, the cellular pathways involved in MDM4 downregulation, which inhibited H1299 cell viability, were elucidated.

Materials and methods

Main reagents

Tan IIA (molecular formula, C19H18O3; >96% high performance liquid chromatography) was obtained from Herbasin (Shenyang) Co., Ltd. (Shenyang, China). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl-sulfoxide (DMSO), actinomycin D, benzo(a)pyrene and DOX) were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). RPMI-1640 and fetal bovine serum (FBS) were supplied by Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The protein isolation kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). MDM4 lentiviral activation particles (cat. no. sc-417855-LAC) and control lentiviral activation particles (cat. no. sc-437282), p73 small interfering RNA (siRNA; cat. no. sc-36167), control siRNA-A (cat. no. sc-37007), and primary antibodies of BCL2 binding component 3 (PUMA; cat. no. sc-374223), anti-p73α (cat. no. sc-7238), anti-MDM2 (cat. no. sc-812), anti-phorbol-12-myristate-13-acetate-induced protein 1 (Noxa; cat. no. sc-56169) and anti-β-actin (cat. no. sc-8432) were all supplied by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and all diluted in Tween buffered saline (1:200) for working solution. Anti-MDM4 (cat. no. ab16058) and anti-inhibitor of apoptosis 3 (IAP3; cat. no. ab21278) antibodies were obtained from Abcam (Cambridge, UK). Anti-Caspase 9 (cat. no. BS6444) and anti-Caspase 3 (cat. no. BS1518) primary antibodies were supplied by Bioworld Technology, Inc. (St. Louis Park, MN, USA). IRDye-conjugated rabbit anti-goat (cat. no. 605-746-002), IRDye-conjugated rabbit anti-mouse (cat. no. 610-446-C46) and IRDye-conjugated mouse anti-rabbit (cat. no. 18-4416-32) secondary antibodies were products of Rockland immunochemicals, Inc. (Limerick, PA, USA). The concentrations of all antibodies were determined according to the manufacturers' protocol.

Cell culture and treatment

In the present study, the H1299 and 16HBE cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The two cell lines were grown in RPMI-1640 standard culture medium containing 10% FBS (v/v), and cultured at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Tan IIA was dissolved in pure grade DMSO to create a 10 mM stock solution. Cells were treated with Tan IIA, or 1.25 µg DOX or 32 µM benzo(a)pyrene at 37°C for 24 h. The mom-treatment group received nothing.

Western blotting

The 80 µM Tan IIA and 1.25 µg DOX treated and untreated cells were harvested for protein extraction using whole cell lysis buffer (1 M Tris-HCl, 5 M NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.05% SDS, 1 mM phenylmethyl sulfonyl fluoride), then centrifuged at 10,000 × g for 30 min at 4°C. Supernatant was collected as whole cell lysate for western blotting analysis. Protein concentrations of the whole cell lysate were examined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China), with FBS as a standard. The whole cell lysates were boiled in SDS Laemmli sample buffer, then 20 µg whole cell lysate/lane was separated by 12% SDS-polyacrylamide gel and subsequently transferred to polyvinylidene fluoride membranes. Following protein transfer, the membranes were blocked at 37°C for 30 min with Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk, then incubated with specific primary antibodies which were dissolved in TBS overnight at 4°C and with the corresponding IRDye-conjugated secondary antibodies (1:5,000 dilution for all) at room temperature for 1 h. The signals of immunoreactive proteins were visualized using the Odyssey Infrared Imaging System and Odyssey version 1.2 software (LI-COR Biosciences, Lincoln, NE, USA). The relative densities of the protein bands were analyzed using Quantity One software (version 4.62; Bio-Rad Laboratories, Inc.). The relative expression of target proteins were normalized to the corresponding intensities of β-actin. Three independent replicates were performed.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA of 32 µM Benzo(a)pyrene- or 80 µM Tan IIA treated and untreated H1299 cells were extracted with TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. First-strand cDNA synthesis (45 cycles) was performed on the 1,000-Series Thermal Cycling Platform with a mixture of random monomers and oligo-dT primers according to the manufacturer's protocol for the First Strand cDNA Synthesis kit (K1612; Thermo Fisher Scientific, Inc.). cDNA amplification was performed at 95°C for 10 sec and 56°C for 30 sec (45 cycles) on a 7900 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), using the Quanti-Fast SYBR Green RT-PCR kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's protocol. All specific primers for the target genes, as well as the housekeeping gene, β-actin, were custom-designed and synthesized by TaKaRa (Dalian, China; sequences not provided). Data was expressed as n-fold change of untreated cells. Each sample mRNA was normalized relative to β-actin using the 2−∆∆Cq method (22).

Analysis of mRNA stability and protein generation

The stability of mRNA was measured by using a standard actinomycin D analysis. Cells were exposed to 5 µg/ml actinomycin D with or without 80 µM Tan IIA for 0, 2 and 4 h, and total RNA was isolated and analyzed as mentioned above. Protein generation was measured using a standard protein-synthesis inhibitor cycloheximide (CHX) as previously described (23). To arrest polyribosome migration, the H1299 cells were treated with 35 µM CHX combined with or without 80 µM Tan IIA for 0, 2 and 4 h, and were then lysed in order to isolate cytoplasmic extracts in a hypotonic lysis buffer containing 10 mM Tris, 1 mM EDTA, and 0.2% Triton X-100. Cell lysates were added to a 15–45 % (w/v) sucrose gradient tube, centrifuged at 4°C, 15,000 × g for 1 h, then the lysate was collected from each gradient tube for western blot analysis.

Transfections

H1299 cells were plated into 6-well plates at a density of 2.5×105 cells/well. Upon reaching 80% confluence, cells were trypsinized and diluted in the RPMI-1640 medium. Transfection of MDM4 lentiviral activation particles (RNA) and control lentiviral activation particles (non-specific RNA) into H1299 cells was performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The p73 siRNA and control siRNA-A transfection was performed using the Entranster-D transfection reagent (Engreen Biosystem Ltd., Beijing, China), following the manufacturer's protocol.

Cytotoxicity assay

The cells were seeded in 96-well plates (~3,000 per well) for 24 h. Total of 80 µM Tan IIA dissolved in DMSO was added to the cells. Following incubation for the 0, 8 and 24 h at 37°C, the culture medium in each well was removed, cells were washed twice with PBS, then incubated with fresh serum-free RPMI-1640 medium containing 10% MTT solution (v/v, concentration: 5 mg/ml) at 37°C for a further 4 h. To solubilize the formazan crystals in the viable cells, the liquid in each well was replaced by 150 µl DMSO. The absorbance of each well was measured at 570 nm using Multi-Detection Microplate Readers (Synergy 2; BioTek Instruments, Inc., Winooski, VA, USA). Data were expressed as a percentage of the control measured in the absence of Tan IIA.

Cell apoptosis assay

An Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit [Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] was used to measure the percentage of apoptotic cells. Treated and untreated cells (~5×105 cells/ml) were centrifuged at 1,000 × g for 5 min at room temperature followed by washing twice with PBS, then the cells were re-suspended in 0.5 ml pre-chilled binding buffer, 5 µl Annexin V-FITC and 10 µl propidium iodide (PI) were added, and the cells were incubated for 30 min at room temperature. Finally, the dead cells were differentiated by FACScan flow cytometry using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed using ModFit software (version 2.0.0; Verity Software House, Inc., Topsham, ME, USA).

Statistical analysis

Data ware presented as the mean ± standard deviation. Comparisons among different groups were analyzed using one-way analysis of variance. If the variation was significant, significant differences between the means of treated and un-treated groups were analysed using Dunnett-t tests. P<0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed using SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA).

Results

Tan IIA suppresses MDM4 expression in H1299 and 16HBE cells

To investigate the effect of Tan IIA (Fig. 1A) on MDM2 and MDM4 expression in H1299 and 16HBE cells, the two cell lines were treated with 40 µM Tan IIA for 24 h. Tan IIA visibly downregulated MDM4, but not MDM2, in the two cell lines studied (Fig. 1B). In addition, Tan IIA downregulated MDM4 protein levels in a dose-dependent manner in H1299 and 16HBE cells (Fig. 1B), even at low concentrations, following treatment for 24 h. The inhibition of MDM4 by Tan IIA occurred ~4 h post-exposure and was followed by a time-dependent reduction during 8–24 h treatment (Fig. 1C).

Tan IIA suppresses MDM4 expression through the repression of MDM4 mRNA synthesis

To understand the mechanism by which Tan IIA downregulates MDM4 expression, MDM4 mRNA expression levels were examined in the p53 deficient H1299 cells following exposure to 80 µM Tan IIA for 1, 2, 4, 8, 16, 24 and 48 h using RT-qPCR. The results demonstrated that Tan IIA significantly downregulated MDM4 mRNA expression 8–48 h post treatment compared with 0 h (P<0.05; Fig. 2A). Next, the influence of mRNA stability on the downregulation of MDM4 mRNA expression in Tan IIA-treated H1299 cells was investigated using a pulse-chase assay and RT-qPCR analysis. There was no difference in MDM4 mRNA stability between the untreated control and the 80 µM Tan IIA-treated group, which indicated that MDM4 mRNA stability was not affected by Tan IIA treatment (Fig. 2B). The involvement of MDM4 protein stabilization in Tan IIA-induced MDM4 inhibition in H1299 cells was then investigated and a standard CHX pulse-chase assay and western blotting were performed. There was no significant difference in MDM4 protein half-life between the untreated control and the 80 µM Tan IIA treated group (Fig. 2C). These results suggested that Tan IIA downregulated MDM4 only through inhibition of MDM4 mRNA synthesis.

Tan IIA-induced MDM4 inhibition upregulates P73α expression

The effect of Tan IIA-inhibited MDM4 expression on P73α expression was then investigated. P73α mRNA levels were measured using RT-qPCR. Treatment with 80 µM Tan IIA did not demonstrate any directly inductive or inhibitory effects on p73 mRNA expression following treatment for 24 h (Fig. 3A). The involvement of Tan IIA in modulation of P73α translation was then investigated, and the H1299 cells were exposed to 80 µM Tan IIA with or without the protein synthesis inhibitor CHX. Tan IIA increased P73α protein expression irrespective of CHX absence or presence, suggesting that Tan IIA does not affect P73α translation (Fig. 3B). The turnover of P73α protein following Tan IIA treatment was measured using a standard pulse-chase assay. The half-life of P73α in the untreated control group was less than <0.5 h, while it was significantly longer in 80 µM Tan IIA treated groups (Fig. 3C), suggesting that the observed upregulation of Tan IIA-induced P73α expression in H1299 cells is only due to post-translational modulation. In addition, the relative levels of MDM4 and P73α were evaluated following treatment of H1299 cells with 80 µM Tan IIA for 8, 16 and 24 h. Tan IIA upregulated P73α levels following the downregulation of MDM4 levels (Fig. 3D).

P73α-induced inhibition of cell viability did not occur in the Tan IIA-treated H1299 cells

p73 has been observed to stimulate transcriptional levels of PUMA and Noxa, which result in mitochondria-dependent apoptosis (24,25). Thus, the expression levels of P73α, MDM4, PUMA and Noxa were examined in H1299 cells following treatment with 80 µM Tan IIA for 24 h using RT-qPCR. Compared with benzo(a)pyrene that induces P73 mRNA (9), Tan IIA failed to upregulate and in fact downregulated P73α, MDM4, PUMA and Noxa mRNA expression levels (Fig. 4A). However, 80 µM Tan IIA treatment for 8, 16 and 24 h visibly increased P73α protein expression, similar to the effect of the 1.25 µg/ml DOX treated group (Fig. 4B), but the increased P73α levels failed to induce an increase in PUMA and Noxa protein expression (Fig. 4B). Although PUMA and Noxa were not upregulated, cell viability was inhibited in the 80 µM Tan IIA-treated group, similar the 1.25 µg/ml DOX-treated group (Fig. 4C).

Knockdown of P73α does not affect Tan IIA-inhibited H1299 cell viability

To further confirm whether P73α exerted a tumor suppressor function in this cell model, P73α was inhibited using siRNA (Fig. 5A). Notably, there was no difference in cell viability between the P73α siRNA group and negative control group following treatment of the cells with 80 µM Tan IIA for 8, 16 or 24 h (Fig. 5B). These results suggested that P73α does not regulate transcriptional activation in response to Tan IIA treatment in this cell model.

Tan IIA inhibits IAP3 in H1299 cells overexpressing MDM4

IAP3 is a known translational target of MDM2 (26) and thus it was hypothesized that Tan IIA-induced downregulation of MDM4 may also affect IAP3 expression. To investigate this, IAP3 protein expression was examined following treatment of H1299 cells with or without 80 µM Tan IIA for 2, 4 and 8 h. IAP3 expression was visibly reduced in the Tan IIA-treated H1299 cells following MDM4 downregulation (Fig. 6A). To further confirm the involvement of Tan IIA-inhibited MDM4 in the reduction of IAP3, H1299 cells were transfected with MDM4 lentiviral activation particles, which express wild-type MDM4, and were termed MDM4-overexpressing H1299 cells. Empty vector control lentiviral activation particles were also transfected and termed the negative control. The cells were then treated with or without 80 µM Tan IIA for 2, 4 and 8 h. A visible reduction of IAP3 expression was detected in the MDM4-overexpressing H1299 cells compared with the negative control H1299 cells (Fig. 6B and C). In addition, no appreciable difference was detected in IAP protein stability between H1299 cells treated with or without Tan IIA (Fig. 6D). These results indicated that Tan IIA suppressed IAP3 expression in H1299 cells overexpressing MDM4 at the transcriptional and translational level.

Tan IIA sensitizes H1299 cells overexpressing MDM4 to DOX-induced apoptosis

To evaluate the effect of Tan IIA on H1299 cells overexpressing MDM4, the cells were exposed to 5–80 µM Tan IIA for 24 h. Treatment with Tan IIA at a concentration of 20–80 µM significantly decreased cell viability in a dose-dependent manner in MDM4-overexpressing and negative control H1299 cells, and the inhibitory effect was stronger in MDM4-overexpressing H1299 cells (Fig. 7A). In addition, the potential synergistic effect of Tan IIA in DOX-induced apoptosis was assessed by treating the H1299 cells overexpressing MDM4 with 80 µM Tan IIA and/or 1.25 µg/ml DOX for 24 h. Tan IIA and DOX significantly induced apoptosis in H1299 cells overexpressing MDM4, and when the same concentrations were given in combination, significantly more cells underwent apoptosis (Fig. 7B). These results suggested that Tan IIA may work in synergy with DOX, and may be able to reverse DOX-resistance in p53-deficient tumor cells.

Discussion

Tan IIA has been widely studied due to its anticancer properties. Tan IIA has previously been reported to inhibit cell proliferation and to induce apoptosis through caspase- and mitochondria-dependent pathways (27). In the present study, the potential effects of Tan IIA on the expression of two p53/p73 negative regulators, MDM2 and MDM4, were investigated in p53-deficient H1299 cells. Tan IIA suppressed MDM4 expression, which resulted in the accumulation of P73α and inhibition of H1299 cell viability. However, the pro-apoptotic function of P73, which induces PUMA and Noxa, were not activated. Tan IIA was also demonstrated to suppress IAP3 expression, which in turn activated Caspase 3 and Caspase 9, as well as induction of cell apoptosis.

Inhibitor-of-apoptosis proteins (IAPs) are the only known endogenous proteins that act to suppress apoptosis via inhibition of initiator and effector caspases (28). Among the IAPs, only IAP3 is a direct inhibitor of caspases, and is the most effective caspase inhibitor compared with the other IAPs (29). IAP3 protein has been demonstrated to bind to and inhibit the activated forms of initiator and effector caspases, which are the enzymes that induce mitochondrial-dependent apoptosis (30). IAP3 expression levels are positively correlated with disease progression and, furthermore, its overexpression is associated with tumorigenesis (31,32). As well as contributing to cancer development, IAP3 has also been reported to contribute to chemotherapy resistance, and inhibiting this protein was demonstrated to efficiently sensitize cells to apoptosis in response to anti-cancer agents (33,34). MDM2 binds to the IAP3 internal ribosome entry site (IRES) RNA through its Really Interesting New Gene (RING) finger domain and upregulates IRES-regulated IAP3 translation, which results in overexpression of IAP3 and resistance to anticancer therapy (26). Since MDM4 also has a RING finger domain through which it is able to interact with MDM2 (35), Tan IIA-induced MDM4 inhibition may affect IAP3 expression. To investigate this, the present study examined whether Tan IIA-induced suppression of MDM4 results in downregulation of IAP3 expression. Tan IIA was demonstrated to suppress IAP3 expression at the transcriptional and translational level, and the inhibition of IAP3 induced by Tan IIA was greater in H1299 cells overexpressing MDM4 than in the negative control H1299 cells.

The inhibition of viability in the H1299 cell model was mainly due to Tan IIA-induced downregulation of IAP3, and this was further confirmed by observation that there was no induction of P73α function and activation of caspase 3 and caspase 9 in these cells. The results demonstrated that, although the suppression of MDM4 induced by Tan IIA upregulated P73α expression in the H1299 cells, the two P73 transcriptional targets, PUMA and Noxa, were not activated. Although the reason why PUMA and Noxa were not activated by Tan IIA-induced upregulation of P73α was not examined, it is possible to hypothesize that the stability of PUMA and Noxa mRNA was decreased by Tan IIA exposure, even though their promoters may have been activated by the upregulation of P73α. The interactions among these proteins require further elucidation. As a Food and Drug Administration-approved chemotherapeutic drug, DOX is used to treat a wide range of types of cancer (36,37). However, clinical use of DOX is limited by its severe side effects, including kidney injury (38). When DOX is given in combination with other antitumor agents, including curcumin or tripeptide, its therapeutic effect is elevated while side effects are relieved, and combination therapy has become the first choice for DOX-based chemotherapy (36,39). The results of the present study further confirmed the results of previous reports (37); demonstrating that Tan IIA-treated H1299 cells overexpressing MDM4 were more sensitive to DOX-induced apoptosis, supporting the potential of Tan IIA as an agent for sensitization of cells to the anti-cancer effect of DOX. This facilitates the avoidance or reduction of the side effects of Tan IIA as it can be effectively used at a lower dose.

In conclusion, the present study demonstrated that Tan IIA exposure resulted in the inhibition of p53 deficient H1299 cell viability through the MDM4-IAP3-caspase signaling pathway, and increased sensitivity of H1299 cells to DOX-induced apoptosis. Thus, it may be possible to use Tan IIA alone or combination with other anti-cancer agents to treat p53-deficient and/or MDM4-overexpressing cancer cells. Further in vivo studies are required to confirm the contribution of Tan IIA to tumor therapy.

Glossary

Abbreviations

Abbreviations:

Tan IIA

Tanshinone IIA

SM

Salvia miltiorrhiza

MDM4

murine double minute 4

DMSO

dimethyl sulfoxide

CHX

cycloheximide

IAP3

inhibitor-of-apoptosis protein 3

DOX

doxorubicin

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February-2018
Volume 17 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Spandidos Publications style
Zu Y, Wang J, Ping W and Sun W: Tan IIA inhibits H1299 cell viability through the MDM4‑IAP3 signaling pathway. Mol Med Rep 17: 2384-2392, 2018
APA
Zu, Y., Wang, J., Ping, W., & Sun, W. (2018). Tan IIA inhibits H1299 cell viability through the MDM4‑IAP3 signaling pathway. Molecular Medicine Reports, 17, 2384-2392. https://doi.org/10.3892/mmr.2017.8152
MLA
Zu, Y., Wang, J., Ping, W., Sun, W."Tan IIA inhibits H1299 cell viability through the MDM4‑IAP3 signaling pathway". Molecular Medicine Reports 17.2 (2018): 2384-2392.
Chicago
Zu, Y., Wang, J., Ping, W., Sun, W."Tan IIA inhibits H1299 cell viability through the MDM4‑IAP3 signaling pathway". Molecular Medicine Reports 17, no. 2 (2018): 2384-2392. https://doi.org/10.3892/mmr.2017.8152