Corrigendum: A novel Chinese herbal and corresponding chemical formula for cancer treatment by targeting tumor maintenance, progression, and metastasis.

[This corrects the article DOI: 10.3389/fphar.2022.907826.].

A Novel Chinese Herbal and Corresponding Chemical Formula for Cancer Treatment by Targeting Tumor Maintenance, Progression, and Metastasis.

We characterized a so-called "heirloom recipe" Chinese herbal formula (temporarily named Formula X) that contains five Chinese medical botanical drugs, Huang-Lian (Coptis chinensis Franch. [Ranunculaceae]), Huang-Qin (Scutellaria baicalensis Georgi [Lamiaceae]), Bai-Wei (Vincetoxicum atratum (Bunge) C. Morren and Decne. [Apocynaceae]), E-Zhu (Curcuma aromatica Salisb. [Zingiberaceae]) and Bai-Zhu (Atractylodes macrocephala Koidz. [Asteraceae]). Formula X inhibited the growth of various cancer cells and decreased the expression levels of a panel of proteins, including CD133, Myc, PD-L1, and Slug, in cancer cells. We further found that the inhibition of growth and protein expression were exerted by Huang-Lian, Huang-Qin, and Bai-Wei (formula HHB), which exhibited the same biological effects as those of Formula X. Furthermore, we selected three active chemicals, berberine, baicalin, and saponin from Huang-Lian, Huang-Qin, and Bai-Wei, respectively, to produce a chemical formulation (formula BBS), which exhibited similar effects on cell growth and protein expression as those induced by formula HHB. Both the formulae HHB and BBS suppressed tumor growth in an animal study. Moreover, they decreased the protein levels of Myc and PD-L1 in tumor cells in vivo. In summary, we established a novel Chinese herbal formula and a chemical formula that targeted three important processes, tumor maintenance (tumor stem cells), progression, and metastasis, and that influenced the response of tumors to host immunosuppression, for the potentially effective treatment of cancer patients.

IκB kinases increase Myc protein stability and enhance progression of breast cancer cells.

BACKGROUND: Both IκB kinase (IKK) complex and oncgenic protein Myc play important roles in cancer progression, including cancer cell invasiveness and metastasis. The levels of Myc is regulated by the phosphorylation of Myc at Thr58 and Ser62. RESULTS: In this study, we show that the expression of Myc is associated with IKKα and IKKß in breast cancers and that Myc is an IKKs substrate. Suppression of IKK activity by either chemical inhibitor or transfection of kinase-dead mutants decreases the phosphorylation of Myc at Ser62 and enhances the degradation of Myc. Consequently, these treatments decrease the tumorigenic and invasive ability of breast cancer cells. Furthermore, doxorubicin, a frequently used anticancer drug in breast cancer, activates IKKs and Myc, thereby increasing invasiveness and tumorigenesis of breast carcinoma MCF7 cells. Inhibition of IKKs prevents these doxorubicin-induced effects. CONCLUSIONS: Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability, and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following chemotherapy to reduce the risk of treatment-induced tumor progression.

Overexpression of B cell-activating factor of TNF family (BAFF) is associated with Helicobacter pylori-independent growth of gastric diffuse large B-cell lymphoma with histologic evidence of MALT lymphoma.

We have recently demonstrated that nuclear expression of BCL10 predicts Helicobacter pylori (HP) independence of early-stage gastric diffuse large B-cell lymphoma (DLBCL) with histologic evidence of mucosa-associated lymphoid tissue (MALT). In this study, we examined the role of B cell-activating factor of TNF family (BAFF) in mediating BCL10 nuclear translocation and HP independence of gastric DLBCL (MALT). We used immunohistochemistry and immunoblotting to measure the expression of BAFF, pAKT, BCL3, BCL10, and NF-kappaB. Transactivity of NF-kappaB was measured by electromobility shift assay. In lymphoma samples from 26 patients with gastric DLBCL (MALT), we detected aberrant expression of BAFF in 7 of 10 (70%) HP-independent and in 3 of 16 (18.8%) HP-dependent cases (P = .015). BAFF overexpression was associated with pAKT expression (P = .032), and nuclear expression of BCL3 (P = .014), BCL10 (P = .015), and NF-kappaB (P = .004). In B-cell lymphoma Pfeiffer cells, BAFF activated NF-kappaB and AKT; the activated NF-kappaB up-regulated BCL10, and the activated AKT caused formation of BCL10/BCL3 complexes that translocated to the nucleus. Inhibition of AKT by LY294002 (a PI3K inhibitor) blocked BCL10 nuclear translocation, NF-kappaB transactivity, and BAFF expression. Our results indicate that autocrine BAFF signal transduction pathways may contribute to HP-independent growth of gastric DLBCL (MALT).

The Aurora kinase inhibitor VE-465 has anticancer effects in pre-clinical studies of human hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and novel therapies are urgently needed. Recently, aberrant expression of Aurora kinases has been reported in various human cancers including HCC. We sought to investigate the potential of a potent and selective Aurora kinase inhibitor, VE-465, for targeted therapy of HCC. METHODS: Cytotoxicity effects of VE-465 were tested in Huh-7 and HepG2 cell lines. Inhibition of Aurora kinase activity was demonstrated by Western blotting and immunofluorescence staining. Mitotic perturbation was visualized by confocal microscopy. Cell cycle profiles and apoptosis were assessed by flow cytometry. In vivo efficacy was determined in nude mice with human HCC xenografts. RESULTS: We demonstrated that VE-465 induced proliferation blockade, histone H3 (Ser10) dephosphorylation, mitotic disturbance, endoreduplication, and apoptosis in Huh-7 and HepG2 cells. We also found that VE-465 suppressed Aurora kinase activity, prevented tumor growth, and induced apoptosis in a Huh-7 xenograft model. CONCLUSIONS: These findings show that VE-465 has potent anticancer effects in human HCC. Inhibitors of Aurora kinases may deserve further exploration as molecular targeted agents against HCC.

NF-kappaB p50 promotes tumor cell invasion through negative regulation of invasion suppressor gene CRMP-1 in human lung adenocarcinoma cells.

Lung adenocarcinoma Cl1-5 cells were selected from parental Cl1-0 cells based on their high metastatic potential. In a previous study, CRMP-1, an invasion suppressor gene, was shown to be suppressed in Cl1-5 cells. However, the regulation of CRMP-1 expression has not been explored. In this study, we showed nuclear factor-kappaB controls CRMP-1 expression. The electromobility shift assay showed that while Cl1-0 cells exhibited low NF-kappaB activity in response to TNF-alpha, an abundance of basal and TNF-alpha-induced NF-kappaB-DNA complex was detected in Cl1-5 cells. Supershift-coupled EMSA and Western blotting of nuclear proteins, however, revealed p50 protein, but not classic p65/p50 heterodimer in the complex. ChIP and EMSA demonstrated that p50 binds to a kappaB site residing between -1753 and -1743 of the CRMP-1 promoter region. Transfection of antisense p50 gene into Cl1-5 cells increased the CRMP-1 protein level and decreased the invasive activity of Cl1-5 cells.

Glucocorticoids enhance cytotoxicity of cisplatin via suppression of NF-{kappa}B activation in the glucocorticoid receptor-rich human cervical carcinoma cell line SiHa.

Glucocorticoids (GCs) are commonly co-administered with cisplatin in the treatment of patients with carcinomas to prevent drug-induced allergic reaction, nausea and vomiting. Although GC receptor (GR) is ubiquitous in carcinoma cells and has been linked to signal transduction pathways pertinent to cell growth and apoptosis, little is known regarding the possible effect of GC on the chemosensitivity of carcinomas. Our previous study demonstrated that dexamethasone (DEX) enhances the cytotoxicity to cisplatin in a GR-rich human cervical carcinoma cell line, SiHa. In this study, we found that this cisplatin cytotoxicity-enhancing effect of DEX correlated well with its effect on abrogating the cisplatin-induced activation of nuclear factor kappa B (NF-kappaB). RU486, a structural homologue of DEX, partially reversed this cytotoxicity-enhancing effect of DEX, a finding consistent with the well-known partial reversing effect of RU486 on DEX-induced NF-kappaB suppression. Furthermore, expression of a dominant-negative truncated IkappaBalpha gene in SiHa cells completely abolished the cisplatin cytotoxicity-enhancing effect of DEX. Our data suggest that the specific action of DEX on GR may enhance the cytotoxicity of cisplatin in selected GR-rich cancer cells by suppressing NF-kappaB activation.

Glucocorticoid receptor expression in advanced non-small cell lung cancer: clinicopathological correlation and in vitro effect of glucocorticoid on cell growth and chemosensitivity.

The current study aimed to immunohistochemically examine the tumor glucocorticoids receptor (GR) expression in patients with advanced non-small cell lung cancer (NSCLC) and to examine the effect of glucocorticoids (GCs) on the in vitro NSCLC cells growth and chemosensitivity. High GR expression was detected in 51% of the tumor specimens. The difference in tumor GR expression was not associated with cell type, gender, age, or stage. The outcome was significantly superior for patients whose tumor showed high GR expression compared to those with either low expression or non-expression. The median progression-free survival was 8.0 versus 5.6 months (p=0.039) and overall survival was 18.1 versus 10.2 months, (p=0.003), respectively. Almost all these patients have received GC as antiemetics or allergic preventive treatment during chemotherapy courses, therefore, the effect of GC on the chemosensitivity in vivo was not evaluable. However, in vitro cytotoxicity assay showed that dexamethasone (DEX) had heterogeneous effects on the growth and chemosensitivity of the NSCLC cell lines. These findings suggest that tumor samples express high levels of GR in about half of the patients with advanced NSCLC, and this high expression of GR may be associated with better outcome. The effect of GC treatment on the chemosensitivity in NSCLC patients remains to be established.

Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death.

We recently reported that exposure of human cervical carcinoma cells to doxorubicin results in extracellular signal-regulated kinase (ERK)2 activation, which in turn phosphorylates p53 on a previously uncharacterized site, Thr55. This study sought to clarify the biological significance of doxorubicin-induced Thr55 phosphorylation. In breast carcinoma MCF7 cells, doxorubicin (300 nM) activated ERK2 and induced phosphorylation of p53 on Thr55 residues. Pretreatment of MCF7 cells with an ERK2 chemical inhibitor, PD98059 or U0126, blocked doxorubicin-induced p53 activation and suppressed phosphorylation of p53Thr55. MCF55a cells were established by transfection of full-length p53 carrying Thr55 mutation (Thr to Ala) into MCF7 cells. Doxorubicin (500 nM) could not induce p53 activation in MCF55a cells, which showed significantly increased drug resistance toward doxorubicin. While the expression of the apoptotic protein, Bax, showed no difference between MCF7 and MCF55a cells, Bcl-2, an antiapoptotic protein, was constitutively expressed in MCF55a cells. The increase of Bcl-2 protein and/or Bcl-2/Bax ratio might at least partly contribute to the drug resistance of MCF55a cells. In summary, our results suggest that phosphorylation of p53Thr55 by ERK2 is important for doxorubicin-induced p53 activation and cell death.

Nuclear expression of BCL10 or nuclear factor kappa B predicts Helicobacter pylori-independent status of early-stage, high-grade gastric mucosa-associated lymphoid tissue lymphomas.

PURPOSE: A high percentage of early-stage, high-grade gastric mucosa-associated lymphoid tissue (MALT) lymphomas remain Helicobacter pylori dependent. t(11;18)(q21;q21), a genetic aberration highly predictive of H. pylori-independent status in low-grade gastric MALT lymphoma, is rarely detected in its high-grade counterpart. This study examined whether nuclear expression of BCL10 or nuclear factor kappa B (NF-kappaB) is useful in predicting H. pylori-independent status in patients with stage IE high-grade gastric MALT lymphomas. PATIENTS AND METHODS: Twenty-two patients who had participated in a prospective study of H. pylori eradication for stage IE high-grade gastric MALT lymphomas were studied. The expression of BCL10 and NF-kappaB in pretreatment paraffin-embedded lymphoma tissues was evaluated by immunohistochemistry and confocal immunofluorescence microscopy. The presence of t(11;18)(q21;q21) was identified by a multiplex reverse transcriptase polymerase chain reaction of the API2-MALT1 chimeric transcript. RESULTS: Aberrant nuclear expression of BCL10 was detected in seven (87.5%) of eight H. pylori-independent and in none of 14 H. pylori-dependent high-grade gastric MALT lymphomas (P <.001). All seven patients with nuclear BCL10 expression had nuclear expression of NF-kappaB, compared with only two of 15 patients without nuclear BCL10 expression (P =.002). As a single variable, the frequency of nuclear expression of NF-kappaB was also significantly higher in H. pylori-independent tumors than in H. pylori-dependent tumors (seven of eight [87.5%] v two of 15 [12.3%]; P =.002). The API2-MALT1 fusion transcript was detected in only one (12.5%) of eight H. pylori-independent lymphomas. CONCLUSION: Nuclear expression of BCL10 or NF-kappaB is highly predictive of H. pylori-independent status in high-grade gastric MALT lymphoma, and coexpression of these two markers in the nuclei is frequent.

Effects of glucocorticoids on the growth and chemosensitivity of carcinoma cells are heterogeneous and require high concentration of functional glucocorticoid receptors.

AIM: To determine how glucocorticoids (GCs) may affect the growth and chemosensitivity of common carcinoma cells. METHODS: The effect of dexamethasone (DEX) on growth and chemosensitivity was assessed in 14 carcinoma cell lines. The function of GC receptors (GR) was assessed by MMTV reporter assay. Overexpression of GR was done by in vitro transfection and expression of a GR-expressing vector. Immunohistochemical stain of tissues and cells were done by PA1-511A, an anti-GR monoclonal antibody. RESULTS: DEX inhibited cell growth of four (MCF-7, MCF-7/MXR1, MCF-7/TPT300, and HeLa), increased cisplatin cytotoxicity of one (SiHa), and decreased cisplatin cytotoxicity of two (H460 and Hep3B) cell lines. The GR content of the seven cell lines affected by DEX was significantly higher than those of the seven cell lines unaffected by DEX (5.2+/-2.5 x 10(4) sites/cell vs 1.3+/-1.4 x 10(4) sites/cell, P = 0.005). Only two DEX-unresponsive cell lines (NPC-TW01 and NPC-TW04) contained high GR amounts in the range (1.9-8.1 x 10(4) sites/cell) of the seven DEX-responsive cell lines. The GR function of NPC-TW01 and NPC-TW04, however, was found to be impaired. The importance of high cellular amount of GR in mediating DEX susceptibility of the cells was further exemplified by GR dose-dependent drug resistance to cisplatin of AGS, a cell line with low GR content and was unaffected by DEX before transfection of GR-expressing vector. Immunohistochemical studies of human cancer tissues showed that 5 of the 45 (11.1%) breast cancer and 43 of the 85 (50.6%) non-small cell lung cancer had high GR contents at the ranges of the GC-responsive carcinoma cell lines. CONCLUSION: The growth and chemosensitivity of human carcinomas with high GR contents may be affected by GC. However, in light of the heterogeneous and even contradictive effects of GC on these cells, routine examination of GR contents of human carcinoma tissues may not be clinically useful until other markers that help predict the ultimate effect of GC on individual patients are identified.

Involvement of nuclear transcription factor-kappa B in low-dose doxorubicin-induced drug resistance of cervical carcinoma cells.

Administration of suboptimal doses of anticancer drugs not only fails to control tumor but often results in increased drug resistance of tumor cells. However, little is known about the effects of transient exposure to a minimally cytotoxic dose of chemotherapy on the development of drug resistance of the tumors. Previous studies have shown that upregulation of drug-exporter proteins (ATP-binding-cassette proteins) may be one of the key mechanisms involved in inducible drug resistance. In this study, we demonstrated that upregulation of NF-kappa B is another possible mechanism. SiHa cells were exposed to low-dose doxorubicin (100 nM; IC(30)) for 3 hr, and then were continuously cultured in drug-free culture media (designated as SiHa/DR cells). SiHa/DR cells at up to 9 passages showed increased resistance to doxorubicin and cross-resistance to cisplatin. Results of quantitative real-time PCR and flow cytometry assay indicated that the increased drug-resistance in SiHa/DR cells was not due to upregulation of drug-exporter proteins or to the decrease of intracellular concentration of anticancer drugs. Both the basal and drug-induced NF-kappa B activity were shown to be increased in SiHa/DR cells by EMSA and NF-kappa B-driven luciferase reporter gene assay. Suppression of NF-kappa B activation by transfection of a dominant negative I kappa B alpha prevented the development of drug resistance, indicating that the upregulated NF-kappa B activity was positively correlated with the low-dose doxorubicin-induced drug resistance. These results suggest that even a transient exposure to a small dose of anticancer drugs may induce drug resistance in some cancer cells via upregulation of NF-kappa B activity.

Increase of the resistance of human cervical carcinoma cells to cisplatin by inhibition of the MEK to ERK signaling pathway partly via enhancement of anticancer drug-induced NF kappa B activation.

In this study, we showed that suppression of the MEK-ERK transduction pathway by a selective inhibitor, 2'-amino-3'-methoxyflavone (PD98059), increased drug resistance of SiHa cells to cisplatin, but not to another common anticancer drug, doxorubicin. The downstream mechanism of this discrepant cellular response was investigated. Both cisplatin and doxorubicin activated nuclear ERK2 and nuclear transcription factor kappa B (NF kappa B) of SiHa cells. However, suppression of the MEK-ERK2 pathway by PD98059 resulted in a further enhancement of cisplatin-induced NF kappa B activation, while no further regulation of NF kappa B was noted in doxorubicin-treated cells. The activation of NF kappa B by cisplatin or doxorubicin was not due to the degradation of cytoplasmic I kappa B alpha, as demonstrated by western blotting. Transfection of a dominant negative I kappa B alpha resulted in a markedly diminished PD98059-induced cisplatin resistance in SiHa cells. Our results suggest that the MEK-ERK signaling pathway plays a role in the chemosensitivity of SiHa cells, and suppression of this pathway increases cisplatin resistance partly via an increase of NF kappa B activation. The mechanism responsible for the discrepant effect of PD98059 on NF kappa B activation and hence the chemosensitivity of SiHa cells towards cisplatin and doxorubicin remains to be investigated.

Basal levels and patterns of anticancer drug-induced activation of nuclear factor-kappaB (NF-kappaB), and its attenuation by tamoxifen, dexamethasone, and curcumin in carcinoma cells.

Nuclear factor-kappaB (NF-kappaB) has been implicated in the development of drug resistance in cancer cells. We systematically examined the baseline levels of NF-kappaB activity of representative carcinoma cell lines, and the change of NF-kappaB activity in response to a challenge with four major anticancer drugs (doxorubicin, 5-fluorouracil, cisplatin, and paclitaxel). We found that the basal level of NF-kappaB activity was heterogeneous and roughly correlated with drug resistance. When challenged with various drugs, all the cell lines examined responded with a transient activation of NF-kappaB which then declined to basal level despite variation in the concentration of the agent and the timing of the treatment. In contrast to tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB in minutes, NF-kappaB activation induced by anticancer drugs usually occurred more than 1hr after stimulation. A gradual increase of total NF-kappaB and its nuclear translocation, and cytoplasmic translocation of nuclear IkappaBalpha and its degradation were involved in this process. In particular, when cells were pretreated with common biologic modulators such as tamoxifen, dexamethasone, and curcumin, the doxorubicin-induced NF-kappaB activation was attenuated significantly. This inhibition may play a role in sensitizing cancer cells to chemotherapeutic drugs. This study has demonstrated that activation of NF-kappaB is a general cellular response to anticancer drugs, and the mechanism of activation appears to be distinct from that induced by TNF-alpha. These observations may have implications for improving the efficacy of systemic chemotherapy for cancer patients.

Doxorubicin activates hepatitis B virus (HBV) replication in HBV-harboring hepatoblastoma cells. A possible novel mechanism of HBV reactivation in HBV carriers receiving systemic chemotherapy.

BACKGROUND: Reactivation of HBV replication is a clinically significant complication in HBV(+) patients receiving chemotherapy. We recently found that nearly half of the HBV reactivation in lymphoma patients occurred within 2 weeks of the first dose of chemotherapy. We hypothesized that mechanisms other than immunosuppression, such as direct stimulation of HBV replication by anticancer drugs, might be involved in this type of HBV reactivation. MATERIALS AND METHODS: 2.2.15 cells, which secrete HBV particles constitutively, were used in the experiments. Real-time quantitative polymerase chain reaction was used to quantitate HBV DNA, and microparticle enzyme immunoassay to measure HBV surface antigen (HBsAg). RESULTS: HBV DNA secretion in culture medium was dose- dependently increased by doxorubicin, one of the most commonly used anticancer drugs for lmphoma. One-hour exposure of cells to 1 microM doxorubicin induced a 15.4+/-5.9-fold and a 3.05+/-0.09-fold increase of HBV DNA and HBsAg on the 4th culture day, respectively. Lamivudine suppressed the doxorubicin-induced increase of HBV DNA. CONCLUSION: Our data suggest that cytotoxic agents may stimulate the replication of HBVand thereby contribute to the reactivation of HBV during systemic chemotherapy. Importantly, this adverse effect of cytotoxic agents may be preventable by co-administration of lamivudine.

Induction of Epstein-Barr virus (EBV) reactivation in Raji cells by doxorubicin and cisplatin.

BACKGROUND: Epstein-Barr virus (EBV) can be activated in B-lymphoid cells to enter the lytic cycle by various kinds of stimuli, including 12-O-tetradecanoylphorbol-1 3-acetate (TPA), butyric acid, calcium ionophore A23187, transforming growth factor-beta and anti-immunoglobulin crosslinking. EBV reactivation has been clinically observed in patients receiving systemic chemotherapy. This study sought in vitro evidence to suggest whether anticancer drugs may directly contribute to the EBV reactivation. MATERIALS AND METHODS: Raji cells, an EBV-containing Burkitt's lymphoma cell line, were used as the experimental model. TPA served as a positive control for chemical induction of EBV reactivation. Expression of the BZLF1 transcript of EBV and its encoded protein, ZEBRA, were examined by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and flow cytometry, respectively. Transactivation activity of ZEBRA was further assessed by a luciferase reporter assay of EBV DR-promoter activity and a flow cytometry assay assessing the endogenous expression of EA-D (BMRFl). RESULTS: Doxorubicin and cisplatin, two commonly used anticancer agents, induced a dose-dependent up-regulation of BZLF1 mRNA and ZEBRA protein. The luciferase reporter activity and the expression of endogenous EA-D protein, also increased by doxorubicin and cisplatin, indicated an up-regulation of the transactivating activity of ZEBRA. CONCLUSION: These data indicate that cytotoxic anticancer drugs may up-regulate the expression and the transactivating activity of BZLF1, and suggest that systemic chemotherapy may be a risk factor for EBV reactivation in patients with EBV-associated malignancies.

Lack of compensatory pAKT activation and eIF4E phosphorylation of lymphoma cells towards mTOR inhibitor, RAD001.

mTOR (mammalian target of rapamycin) inhibitors were recently found to be effective in the treatment of various human non-Hodgkin's lymphomas (NHLs). We recently reported that RAD001, an mTOR inhibitor, suppressed the growth of lymphoma cells at concentrations much lower than those required for carcinomas. However, the basis for the enhanced sensitivity to RAD001 is unknown. Seven aggressive NHL cell lines and seven carcinoma cell lines were used in this study. Cell cycle was analysed by flow cytometry. pAKT (phosphorylated AKT) (Ser(473) and Thr(308)), p-p70S6K, p-4E-BP1, p-mTOR, p-eIF4E (phosphorylated eIF4E), cyclin A, cyclin E, cyclin D3, c-Myc and insulin receptor substrate-1 (IRS-1) protein expression were assessed by immunoblotting. The PI3K/AKT/mTOR (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin) signalling pathway was constitutively expressed in all seven lymphoma cell lines. RAD001 down-regulated p-mTOR, p-p70S6K, p-4E-BP1, cyclin A, cyclin E, cyclin D3, and c-Myc, but did not affect IRS-1. In parallel with RAD001-induced inhibition of cell viability, a dose- and schedule- dependent down-regulation of pAKT and p-eIF4E expressions was demonstrated. In contrast, a compensatory activation of pAKT and p-eIF4E, was observed in seven carcinoma cells. These findings indicate that the basis for enhanced activity of mTOR inhibitors in NHL may be the lack of compensatory activation of AKT and eIF4E phosphorylation in lymphoma cells.

Combinations of mTORC1 inhibitor RAD001 with gemcitabine and paclitaxel for treating non-Hodgkin lymphoma.

Single-agent mammalian target of rapamycin complex 1 (mTORC1) inhibitors have recently been reported as effective salvage treatment in non-Hodgkin lymphoma (NHL). The combined effect of mTORC1 inhibitor, RAD001, with chemotherapeutic agents used for relapsed or refractory NHL was examined. Synergistic interactions were observed for RAD001 plus gemcitabine or paclitaxel in six NHL cell lines; enhanced gemcitabine- and paclitaxel-induced caspase-dependent apoptosis associated with down-regulation of mTOR signaling was detected. Synergistic interactions were also observed with RAD001 plus gemcitabine and paclitaxel. In conclusion, synergistic cytotoxicity was observed with RAD001 plus gemcitabine and paclitaxel in NHL cells. Combination therapy with these three drugs should be examined in patients with refractory or relapsed NHL.

Induction of DNA damage-inducible gene GADD45beta contributes to sorafenib-induced apoptosis in hepatocellular carcinoma cells.

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45ß (GADD45ß), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45ß expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45ß mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45ß induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45ß induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45ßor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45ß promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45ß induction by sorafenib. Together, our findings suggest that GADD45ß induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.

Bortezomib overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells in part through the inhibition of the phosphatidylinositol 3-kinase/Akt pathway.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.