Activation of CD40, a member of the tumor necrosis factor receptor (TNF-R) family, results in growth inhibition or apoptosis in some tumor cells, making CD40 a potential antitumor therapeutic target. Although it is known that CD40 is able to induce tumor necrosis factor-alpha (TNF-α) secretion and potentiate cisplatin’s anticancer activity, whether TNF-α induction is involved in sensitizing cisplatin by CD40 has not been addressed. In this report, we provide evidence substantiating an important role of autocrine TNF-α in potentiation of cisplatin-induced apoptosis by recombinant soluble CD40 ligand (rsCD40L) in different human cancer cell lines. Activation of CD40 by rsCD40L induces two phases of autocrine TNF-α: the rapid early phase involving p38 MAP kinase and the robust and persistent late phase through enhanced tnf-α gene transcription. Blocking TNF-α with either a specific TNFR1 siRNA or a neutralizing anti-TNF-α antibody dramatically attenuated the potentiation effect of rsCD40L on cisplatin-induced cancer cell death. These results reveal an important role of TNF-α induction in CD40’s chemosensitization activity and suggest that modulating TNF-α autocrine from cancer cells is an effective option for increasing the anticancer value of chemotherapeutics such as cisplatin. (Cancer Sci 2012; 103: 197–202)
CD40, a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily, is expressed on numerous types of cells including immunocytes, fibroblasts, endothelial and epithelial cells. The ligation of CD40 with its cognate ligand (CD40L, CD154), a type II integral membrane protein with homology to TNF mainly expressed on activated T cells, plays a central role in the regulation of adaptive immunity.(1,2) CD40 and CD40L interaction in immune cells results in potent activation of multiple pathways, including Jun N-terminal protein kinase (JNK), p38 mitogen-activated protein (MAP) kinase, extracellular signal-related kinase (ERK) and transcription factor nuclear factor κB (NF-κB), accompanied by induction of various cytokines (e.g. interleukin (IL)-1, IL-4, IL-6, IL-8, IL-10, IL-12, GM-CSF, TNF-α and Regulated upon Activation, Normal T-cell Expressed, and Secreted [RANTES]) and upregulation of adhesion and co-stimulatory molecules (including ICAM-1, CD23, CD80, CD86 and CD106).(3–6)
CD40 has also been demonstrated to be expressed on the surfaces of a variety of malignant cells (including different carcinoma, melanoma, multiple myeloma, lymphoma).(7) Although there are reports showing that CD40 ligation on tumor cells results in increased cell survival and activation,(8,9) a growing number of studies have indicated that ligation of cell surface CD40 on certain tumor cells might convey a pro-apoptotic or inhibitory signal.(10–14) The ability of CD40 to mediate apoptosis in tumor cells is intriguing, because CD40, unlike other members of the TNFR superfamily, does not contain a death domain in its cytoplasmic terminus. Instead, it has been reported that CD40 ligation induces apoptosis of tumor cells through activation of cytotoxic ligands of the TNF superfamily, including Fas ligand, TNF-related apoptosis-inducing ligand (TRAIL) and TNF-α, thus causing cell death through autocrine or paracrine mechanisms.(10) CD40 ligation on tumor cells also sensitizes cells to apoptosis induced by a variety of agents, including TRAIL, Fas-ligand and chemotherapeutic drugs.(12,15,16) In addition to directly suppressing tumor cell growth and inducing tumor cell apoptosis, it has been well demonstrated that CD40 activation could potentiate antitumor immunity in vitro as well as in vivo.(17–19)
Although it is known that CD40 is able to induce TNF-α secretion and potentiate cisplatin’s anticancer activity, whether TNF-α induction is involved in sensitizing cisplatin’s ability to kill cancer cells by CD40 has not been elucidated. In this study we demonstrate that CD40 activation sensitizes human cancer cells to cisplatin-induced apoptosis, which is associated with CD40-induced autocrine TNF-α achieved through enhancing both TNF secretion and tnf-α gene transcription. Blocking TNF-α dramatically attenuated the potentiation effect of rsCD40L on cisplatin-induced cancer cell death. These results reveal an important role of TNF-α induction in CD40’s chemosentization activity and suggest that modulating TNF-α autocrine from cancer cells is an effective option for increasing the anticancer value of chemotherapeutics such as cisplatin.
Materials and Methods
Reagents. Recombinant soluble CD40 ligand (rsCD40L), human TNF-α and TNF-α neutralizing antibody were from PeproTech (Rocky Hill, NJ, USA). Cisplatin and actinomycin D (Act-D) were from Sigma (St Louis, MO, USA). Caspase inhibitor Z-VAD-FMK, IETD-CHO, LEHD-CHO, TNF-α converting enzyme (TACE) inhibitor-1 (TAPI-1), ERK inhibitor (328006) and IKK inhibitor II were from Calbiochem (La Jolla, CA, USA). JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190 were from Tocris Bioscience (Ellisville, MO, USA). Small interfering RNA (siRNA) for TNFR1 and negative control siRNA were from Guangzhou RiboBio Co. (Guangzhou, China). siRNA transfection reagent was from Polyplus transfection (Illkirch, France). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). The following antibodies were used for western blot: anti-active-caspase-3, anti-caspase-8 and anti-poly (ADP-ribose) polymerase (PARP) (BD bioscience, San Diego, CA, USA); anti-CD40 and anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Fas (Stressgen Biotechnologies, Victoria, Canada); anti-DR5 (eBioscience, San Diego, CA, USA); and anti-β-actin (ProteinTech Group, Chicago, IL, USA).
Cell culture. An ovarian cancer cell line SKOV3, a cervical cancer cell line Siha and a non-small-cell lung cancer cell line A549 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 or DMEM with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1 mmol/L glutamate, 100 units/mL penicillin and 100 μg/mL streptomycin under standard incubator condition (37°C, 5% CO2).
Cell death and apoptosis assay. Cell death was quantitively determined using a lactate dehydrogenase (LDH) release-base cytotoxicity detection kit (Promega, Madison, WI, USA) as described previously.(20) All experiments were repeated three to five times and the average is shown in each figure. Flow cytometry was used to detect apoptosis by using an Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech, Nanjing, China) as described previously.(21)
Western blot. Cells were treated as indicated in the figure legend and lysed in M2 buffer as described previously.(20) Equal amounts of protein extracts were resolved by SDS-PAGE and the proteins of interest were probed using western blot. The proteins were visualized using enhanced chemiluminescence according to the manufacturer’s instructions (Millipore, Billerica, MA, USA). Each experiment was repeated at least three times and representative results are shown in each figure.
Enzyme-linked immunosorbent assay (ELISA) and fluorescent immunocytostaining. Autocrine TNF-α in culture medium was measured by ELISA using an ELISA kit from NeoBioscience Technology Co., Ltd (Shenzhen, China) following the manufacturer’s instructions. To detect the expression of CD40 on the cell membrane, fluorescent immunocytostaining was performed by incubation with rabbit anti-CD40 antibody followed by staining with FITC-conjugated goat anti-rabbit IgG antibody. Representative images of cells were taken with a fluorescence microscope.
Knockdown of TNF-α receptor 1 (TNFR1) by RNA interference. Small interfering RNA targeting TNFR1 and the silencer negative control siRNA were transfected with siRNA transfection reagent (polyplus transfection) following the manufacturer’s instructions. Forty-eight hours after transfection, cells were treated with rsCD40L or cisplatin individually or in combination and then followed by LDH release-based cell death assay.
Quantitative real-time RT-PCR. Total RNA was extracted with TRIzol reagent and reverse transcribed to cDNA using a reverse transcription kit (TransGen Biotech, Beijing, China). The quantitative real-time PCR was carried out using a SYBR green I Real-Time Quantitative PCR kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. The primers used were as follows: TNF-α, forward primer 5′-AGCCCATGTTGTAGCAAACC-3′ and reverse primer 5′-TGAGGTACAGGCCCTCTGAT-3′; and β-actin, forward primer 5′-CCAGCCTTCCTTCCTGGGCAT-3′ and reverse primer 5′-AGGAGCAATGATCTTGATCTTCATT-3′. The PCR mixture was denatured at 94°C for 2 min and amplification was run at 94°C for 30 s, 60°C for 30 s and 68°C for 30 s for 40 cycles. The relative expression value of the TNF-α gene in each sample was determined by standard curve, and then normalized to the relative expression value of β-actin. The results were presented as fold of TNF-α/β-actin mRNA and expressed as mean ± SD.
Statistical analysis. All numerical data were presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was analyzed by paired Student’s t test using the SPSS statistics software package (IBM SPSS, Chicago, IL, USA) and P < 0.05 was considered statistically significant.
CD40 ligation potentiates cisplatin-induced cytotoxicity in cancer cells. We first confirmed that CD40 was nicely expressed in SKOV3 cells, Siha cells and A549 cells as determined using western blotting (Fig. 1a). Immunofluorescence staining also showed typical membrane localization of CD40 molecules (Fig. 1b). rsCD40L, a soluble CD40L comprising the receptor binding TNF-like domain of CD40L, was then used to examine the effect of CD40 activation on cisplatin-induced cancer cell death. SKOV3 cells were insensitive to rsCD40L-induced cytotoxicity as marginal cell death (around 10%) was detected at the highest evaluated dose of rsCD40L (2 μg/mL). A dose-dependent synergistic cytotoxicity, which was evaluated using combination index (CI) analysis as described previously(22) (Supporting Information Table S1), was detected when cells were treated with 10 μmol/L cisplatin and increasing concentrations of rsCD40L. A similar dose-dependent synergism was found with a fixed rsCD40L dose (0.5 μg/mL) and increasing concentrations of cisplatin (5–15 μmol/L) (Fig. 1c,d). The synergistic effect was also achieved in Siha and A549 cells (Fig. 1e), although the concentrations of reagents used in these cells were higher than that used in SKOV3 cells. In addition, rsCD40L also sensitized cancer cells to doxorubicin-, etopside- and paclitaxel-induced cytotoxicity (Supporting Information Fig. S1).
rsCD40L and cisplatin co-treatment enhances apoptosis in cancer cells. To determine which mode of cell death is induced by rsCD40L and cisplatin co-treatment, we first examined apoptosis in SKOV3 cells by flow cytometry after annexin V and propidium iodide (PI) staining. Both early apoptotic cells (annexin V+/PI− staining) and late apoptotic cells (annexin V+/PI+ staining) were significantly increased in cells co-treated with rsCD40L and cisplatin, indicating that the enhanced cell death is mainly due to apoptosis (Fig. 2a). The enhanced apoptosis in co-treated SKOV3 cells was further confirmed by the activation of caspase, activation of caspase 3 and the cleavage of the caspase-3 substrate PARP, both of which were potentiated in the co-treated cells (Fig. 2b). Consistently, the pan-caspase inhibitor z-VAD-FMK profoundly inhibited synergistic cell death in rsCD40L and cisplatin co-treated SKOV3 cells, Siha cells and A549 cells, respectively (Fig. 2c–e). As expected, the caspase-9 inhibitor LEHD-CHO partially protected cells from death because cisplatin mainly activates the intrinsic apoptosis pathway (Fig. 2c). Interestingly, the caspase-8 inhibitor IETD-CHO also partially inhibited the potentiation effect of rsCD40L (Fig. 2c), which is consistent with a modest enhancement of caspase 8 activation observed in rsCD40L and cisplatin co-treated cells (Fig. 2b), suggesting activation of the extrinsic apoptosis pathway. Therefore, activation of both extrinsic and intrinsic apoptosis pathways might underlie the main mechanism for the synergistic cell death induced by co-treatment of cisplatin and rsCD40L.
CD40 activation induces TNF-α autocrine in cancer cells. Next, we proceeded to investigate how rsCD40L triggers activation of the extrinsic apoptosis pathway. The expression of death receptors, TNFR1, DR5 and Fas, remained unchanged in SKOV3 cells after rsCD40L and cisplatin cotreatment (Fig. 3a), suggesting that it is unlikely that CD40-mediated potentiation of cisplatin-induced cytotoxicity is mediated through increasing expression of these death receptors. We then examined if rsCD40L affects secretion of the death ligand cytokines. Both TRAIL and Fas ligand could hardly be detected in the medium from cells either cultured with or without rsCD40L (data not shown). However, rsCD40L robustly induced TNF-α secretion to the culture media, which started as early as 5 min and reached a peak level at 24 h after rsCD40L treatment (Fig. 3b). Interestingly, it appeared that there were two phases of TNF-α induction: a rapid response in the first 30 min (Fig. 3b, inset) and a persistent increase from 60 min to 24 h (Fig. 3b). Cisplatin exerted no detectable effect on basal or rsCD40L-induced TNF-α secretion (Fig. 3c). rsCD40L-induced TNF-α secretion was also strongly detected in Siha and A549 cells (Fig. 3d). These results provided clear evidence showing that CD40 activation triggers TNF-α autocrine in cancer cells.
Autocrine TNF-α is critical for CD40-mediated potentiation of cisplatin-induced cytotoxicity. To determine the role of autocrine TNF-α in CD40-mediated potentiation of cisplatin-induced cytotoxicity, we transfected SKOV3 cells with TNFR1 siRNA to inhibit TNFR1 expression to block TNF-α-induced apoptotic signaling (Fig. 4a, inset). TNFR1 siRNA dramatically inhibited rsCD40L and cisplatin-induced cell death (Fig. 4a). Additionally, a TNF-α neutralizing antibody that blocks TNF-α binding to its receptor also significantly suppressed enhanced cell death in both SKOV3 and Siha cells (Fig. 4b,c). The potentiation of cisplatin-induced cell death by TNF-α was further substantiated by co-treating the cells with cisplatin plus recombinant TNF-α, which showed a dose-dependent synergistic effect in all cell lines (Fig. 5a–c). Altogether, these results suggest that autocrine TNF-α is critical for CD40-mediated potentiation of cisplatin-induced cytotoxicity.
CD40 activation induces TNF-α autocrine through both increased TNF-α secretion and TNF-α mRNA expression. To determine whether TNF-α autocrine induced by CD40 ligation results from increased gene transcription, real-time quantitative RT-PCR was performed. As shown in Figure 6a, TNF mRNA expression was significantly increased by rsCD40L treatment, which could be detected at 15 min and reached a peak at 30 min post-treatment. Because TNF-α autocrine could be measured 5 min after rsCD40L treatment (at which time there was no TNF-α mRNA increase), it is unlikely the CD40 activation-mediated autocrine of TNF-α is solely achieved through increasing TNF-α mRNA expression. Consistent with this hypothesis, pretreatment of SKOV3 cells with the transcription inhibitor actinomycin D, which effectively suppressed TNF-α mRNA expression (Fig. 6b), only inhibited the late phase of TNF-α secretion (Fig. 6c) and had no detectable effect on the early phase of TNF-α secretion (Fig. 6c, inset). These results suggest that the early phase of TNF-α secretion is through enhanced secretion of TNF-α from the cell membrane and intracellular space to the culture media while the late phase is through transcription-driven TNF-α expression. Interestingly, TNF-α secretion in the early phase could be partly suppressed by the TACE inhibitor TAPI-1. In addition, the p38 MAPK inhibitor, but not the inhibitors for JNK, ERK or NF-κB, strongly suppressed early phase TNF-α secretion (Fig. 6d). These results suggest that TACE and p38 MAPK might be involved in the early phase TNF-α secretion induced by rsCD40L.
In the present study we found that autocrine TNF-α induced by CD40 activation plays an important role in CD40-mediated potentiation of cisplatin-induced apoptosis in a number of human cancer cell lines derived from different organs. The results show that activation of CD40 by rsCD40L induces two phases of TNF-α autocrine: the rapid early phase involving p38 MAP kinase and the robust and persistent late phase through enhanced tnf-α gene transcription. Blocking TNF-α with specific siRNA targeting TNFR1 or a neutralizing anti-TNF-α antibody dramatically attenuated the potentiation effect of rsCD40L on cisplatin-caused cancer cell death. These results strongly suggest that CD40 activation mediates sensitization on cisplatin-based cancer chemotherapy through induction of TNF-α autocrine and modulating TNF-α autocrine from cancer cells is an effective option to increase the anticancer value of chemotherapeutics such as cisplatin.
The main finding of the present study is that autocrine TNF-α induced by CD40 activation is important for potentiating cisplatin-induced cancer cell death. The fact that the caspase-8 inhibitor effectively alleviated the cytotoxic potentiation implies that the TNF-α-induced extrinsic apoptotic pathway plays a major role in the synergy of cancer cell death. Indeed, because CD40 does not have a death domain in its cytoplasmic region, autocrine TNF-α appears to be an intriguing mechanism for CD40 to activate the extrinsic apoptotic pathway. Although it has been reported that CD40 induced cancer cell apoptosis through activation of TNF-α,(10) and rsCD40L therapy exhibited an augmented antitumor effect with cisplatin by increasing expression of FasL in an ovarian tumor xenograft SCID mice model,(15) to our knowledge this is the first report showing that CD40 activation enhances cisplatin-caused cancer cell death through inducing autocrine of TNF-α.
The induction of TNF-α autocrine in cancer cells by CD40 activation is likely to be in two phases: the early phase is achieved through enhancing TNF-α secretion and the late phase is through increasing tnf-α gene transcription. The TNF-α mRNA expression level was significantly increased at 30 min after rsCD40L treatment and transcription inhibitor actinomycin D completely blocked the late phase of TNF-α secretion providing strong evidence showing that the late phase of TNF-α secretion was mainly mediated through gene transcription. The early phase of TNF-α secretion likely involves TACE, a disintegrin and metalloprotease (ADAMs) family member that catalyzes the release of TNF-α by shedding the membrane-anchored TNF-α precursor,(23) and p38 MAPK. The detailed mechanisms by which CD40 activation regulates TNF-α autocrine are undoubtedly worthy of further study.
Altogether, our results strongly suggest an important role for autocrine TNF-α in CD40-mediated potentiation of cisplatin-induced apoptosis in a number of human cancer cells. The combination of rsCD40L and cisplatin, the most widely used frontline chemotherapeutic drug, in inducing cancer cell death makes CD40 a good candidate for sensitizing cisplatin-based cancer therapy. Data from this study further suggest that modulation of TNF-α autocrine from cancer cells is an effective option for increasing anticancer value of chemotherapeutics such as cisplatin.
This study was supported in part by grants from National Natural Science Foundation of China (30772539 and 30973403), a grant from the Young Scientist Fund of the Science and Technology Department of Sichuan Province, China (2010JQ0012), and a grant from the Scientific Research Foundation for the Returned Overseas Chinese Scholar, State Education Ministry of China.
There is no potential conflict of interest or financial dependence regarding this publication.