Liposomes targeting the cancer cell-exposed receptor, claudin-4, for pancreatic cancer chemotherapy.

BACKGROUND: Claudin-4 (CLDN4), a tight junction protein, is overexpressed in several types of cancer, and is considered a biomarker for cancer-targeted treatment. CLDN4 is not exposed in normal cells, but becomes accessible in cancer cells, in which tight junctions are weakened. Notably, surface-exposed CLDN4 has recently been found to act as a receptor for Clostridium perfringens enterotoxin (CPE) and fragment of CPE (CPE17) that binds to the second domain of CLDN4. METHODS: Here, we sought to develop a CPE17-containing liposome that targets pancreatic cancers through binding to exposed CLDN4. RESULTS: Doxorubicin (Dox)-loaded, CPE17-conjugated liposomes (D@C-LPs) preferentially targeted CLDN4-expressing cell lines, as evidenced by greater uptake and cytotoxicity compared with CLDN4-negative cell lines, whereas uptake and cytotoxicity of Dox-loaded liposomes lacking CPE17 (D@LPs) was similar for both CLDN4-positive and negative cell lines. Notably, D@C-LPs showed greater accumulation in targeted pancreatic tumor tissues compared with normal pancreas tissue; in contrast, Dox-loaded liposomes lacking CPE17 (D@LPs) showed little accumulation in pancreatic tumor tissues. Consistent with this, D@C-LPs showed greater anticancer efficacy compared with other liposome formulations and significantly extended survival. CONCLUSIONS: We expect our findings will aid in the prevention and treatment of pancreatic cancer and provide a framework for identifying cancer-specific strategies that target exposed receptors.

Dual Effect of Chemo-PDT with Tumor Targeting Nanoparticles Containing iRGD Peptide.

Nanotechnology, including self-aggregated nanoparticles, has shown high effectiveness in the treatment of solid tumors. To overcome the limitations of conventional cancer therapies and promote therapeutic efficacy, a combination of PDT and chemotherapy can be considered an effective strategy for cancer treatment. This study presents the development of tumor-targeting polysialic acid (PSA) nanoparticles for chemo-PDT to increase the cellular uptake and cytotoxic effect in cancer cells. Chlorin e6 (Ce6), a photosensitizer, and the iRGD peptide (sequence; cCRGDKGPDC) were conjugated to the amine of N-deacetylated PSA. They generate reactive oxygen species (ROS), especially singlet oxygen (1O2), and target integrin αvß3 on the cancer cell surface. To offer a chemotherapeutic effect, doxorubicin (Dox) was assembled into the core of hydrophobically modified PSA by connecting it with Ce6; this was followed by its sustained release from the nanoparticles. These nanoparticles are able to generate ROS under 633 nm visible-light irradiation, resulting in the strong cytotoxicity of Dox with anticancer effects in HCT116 cells. PSA nanoparticles with the dual effect of chemo-PDT improve conventional PDT, which has a poor ability to deliver photosensitizers to cancer cells. Using their combination with Dox chemotherapy, rapid removal of cancer cells can be expected.

Bakuchiol, main component of root bark of Ulmus davidiana var. japonica, inhibits TGF-ß-induced in vitro EMT and in vivo metastasis.

Cancer is a second leading cause of death worldwide, and metastasis is the major cause of cancer-related mortality. The epithelial-mesenchymal transition (EMT), known as phenotypic change from epithelial cells to mesenchymal cells, is a crucial biological process during development. However, inappropriate activation of EMT contributes to tumor progression and promoting metastasis; therefore, inhibiting EMT is considered a promising strategy for developing drugs that can treat or prevent cancer. In the present study, we investigated the anti-cancer effect of bakuchiol (BC), a main component of Ulmus davidiana var. japonica, in human cancer cells using A549, HT29 and MCF7 cells. In MTT and colony forming assay, BC exerted cytotoxicity activity against cancer cells and inhibited proliferation of these cells. Anti-metastatic effects by BC were further confirmed by observing decreased migration and invasion in TGF-ß-induced cancer cells after BC treatment. Furthermore, BC treatment resulted in increase of E-cadherin expression and decrease of Snail level in Western blotting and immunofluorescence analysis, supporting its anti-metastatic activity. In addition, BC inhibited lung metastasis of tail vein injected human cancer cells in animal model. These findings suggest that BC inhibits migration and invasion of cancers by suppressing EMT and in vivo metastasis, thereby may be a potential therapeutic agent for treating cancers.

Targeting of claudin-4 by Clostridium perfringens enterotoxin-conjugated polysialic acid nanoparticles for pancreatic cancer therapy.

Despite recent advances in chemotherapy, pancreatic cancer remains a leading cause of cancer-related deaths. Moreover, although targeted therapy has shown promising therapeutic efficacy in many types of cancers, no such effective targeting strategy for treatment of pancreatic cancer, which is in desperate need for new treatment approaches. Here, we developed claudin-4-targeting Clostridium perfringens enterotoxin (CPE) peptide-conjugated polysialic acid nanoparticles (C-SNPs) for pancreatic cancer-targeted therapy. Doxorubicin-loaded C-SNPs (DOX-C-SNPs) higly accumulated in the targeted pancreatic cancer via enhanced peameability and retention (EPR) effect, targeting claudin-4 in pancreatic cancer that becomes superficially exposed owing to the disruption of tight junctions. Notably, DOX-C-SNP accumulation in the non-targeted, normal pancreas was significantly reduced because of hindered access to claudin-4 in tight junctions. As a result, DOX-C-SNPs substantially suppressed tumor growth in an orthotopic pancreatic cancer model while exerting minimal toxicity against non-targeted, normal tissues. Collectively, these findings indicate that claudin-4-targeting DOX-C-SNPs may have promise in treating pancreatic cancers through targeting of exposed claudin-4.

Amentoflavone, active compound of Selaginella tamariscina, inhibits in vitro and in vivo TGF-ß-induced metastasis of human cancer cells.

Epithelial mesenchymal transition (EMT) is a well-known and important step in metastasis and thus can be a key target in cancer treatment. Here, we tested the EMT inhibitory actions of Selaginella tamariscina and its active component, amentoflavone (AF). EMT was examined in vitro using wound-healing and invasion assays and by monitoring changes in the expression of the EMT-related proteins, E-cadherin, Snail, and Twist. Metastasis was examined in vivo using SCID mice injected with luciferase-labeled A549 cells. We confirmed that aqueous extracts of S. tamariscina (STE) and AF inhibited EMT in human cancer cell lines. We found that STE and AF at nontoxic concentrations exerted remarkable inhibitory effects on migration (wound healing assay) and invasion (Transwell assay) in tumor necrosis factor (TGF)-ß-treated cancer cells. Western blotting and immunofluorescence imaging show that AF treatment also restored E-cadherin expression in these cells compared to cells treated with TGF-ß only. Suppression of metastasis by AF was investigated by monitoring migration of tail-vein-injected, circulating A549-luc cells to the lungs in mice. After 3 wk, fewer nodules were observed in mice co-treated with AF compared with those treated with TGF-ß only. Our findings indicate that STE and AF are promising EMT inhibitors and, ultimately, potentially potent antitumor agents.

Hypoxia-responsive folic acid conjugated glycol chitosan nanoparticle for enhanced tumor targeting treatment.

Hypoxia is a characteristic feature of various ischemic diseases, including cancer. This study describes the development of glycol chitosan nanoparticles, hydrophobically modified with 4-nitrobenzyl chloroformate and folic acid (FA), that can specifically release drugs under hypoxic conditions. This hypoxia-responsive glycol chitosan nanoparticle conjugated with FA (HRGF) possesses tumor-targeting properties by virtue of conjugated FA and is able to release drugs in a nitroreductase (NTR)-dependent manner because its structure is cleaved by NTR under hypoxic conditions. HRGF nanoparticles showed improved in vivo cancer-targeting ability compared with HRG nanoparticles without FA. In vitro drug release profiles revealed that doxorubicin (DOX)-loaded HRGF (D@HRGF) nanoparticles showed rapid release under hypoxia conditions than normoxic conditions. In vitro cytotoxicity tests and microscopic observations showed that D@HRGF nanoparticles were more toxic towards hypoxic cells than normoxic cells, and that the release of DOX was more effective in hypoxia than normoxia. In vivo, D@HRGF nanoparticles showed more effective antitumor activity in mice compared with D@HRG and free DOX. Collectively, these results show that HRGF nanoparticles function as an effective drug-delivery system in hypoxic conditions. Moreover, these hypoxia-responsive nanoparticles would be effective not only in cancer, but also in other ischemic diseases.

Hispolon as an inhibitor of TGF-ß-induced epithelial-mesenchymal transition in human epithelial cancer cells by co-regulation of TGF-ß-Snail/Twist axis.

Hispolon (HPL), isolated from Phellinus linteus, has been used to treat various types of pathology, including inflammation, gastroenteric disorders, lymphatic diseases and numerous cancer subtypes. HPL has previously been reported to demonstrate a significant therapeutic efficacy against various types of cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder and gastric cancer cells. However, its potential role in the epithelial-mesenchymal transition (EMT) has not been demonstrated. The present study investigated the effects of HPL on the EMT. Transforming growth factor ß (TGF-ß) induced enhanced cell migration and invasion, EMT-associated phenotypic changes. In the present study, HPL recovered the reduction of E-cadherin expression level in TGF-ß treated cancer cells, which was regulated by the expression of Snail and Twist. HPL downregulated Snail and Twist, an effect that was enhanced by TGF-ß. These findings provide novel evidence that HPL suppresses cancer cell migration and invasion by inhibiting EMT. Therefore, HPL may be a potent anticancer agent, inhibiting metastasis.

Piperlongumine inhibits TGF-ß-induced epithelial-to-mesenchymal transition by modulating the expression of E-cadherin, Snail1, and Twist1.

Cancer is a life-threatening disease, and the occurrence of metastasis, which increases the lethality of primary tumors, is increasing. The epithelial-to-mesenchymal transition (EMT) is a biological process by which epithelial cells lose cell-cell adhesion properties and acquire mesenchymal properties, including motility and invasiveness. EMT is considered an early stage of metastasis; therefore, inhibiting EMT may be an effective anticancer therapy. In the present study, the antimetastatic effect of piperlongumine (PL) was assessed in human cancer cells. PL is a single component isolated from long pepper (Piper longum) and it has been studied for its antibacterial, antiangiogenic, and antidiabetic activities. Migration assays (wound healing assay) and transwell invasion assays showed that PL inhibited the migration and invasion of cancer cells. Western blotting and immunofluorescence imaging showed that TGF-ß upregulated the transcription factors Snail1 and Twist1 and downregulated E-cadherin, a marker of epithelial cells, inducing EMT. PL might inhibit TGF-ß-induced EMT by downregulating Snail1 and Twist1 and upregulating E-cadherin in cancer cells. In summary, PL might inhibit TGF-ß-induced EMT, suggesting that it is a promising anticancer agent.

Hydrophobically modified polysaccharide-based on polysialic acid nanoparticles as carriers for anticancer drugs.

This study presented the development of hydrophobically modified polysialic acid (HPSA) nanoparticles, a novel anticancer drug nanocarrier that increases therapeutic efficacy without causing nonspecific toxicity towards normal cells. HPSA nanoparticles were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling between N-deacetylated polysialic acid (PSA) and 5ß-cholanic acid. The physicochemical characteristics of HPSA nanoparticles (zeta-potential, morphology and size) were measured, and in vitro cytotoxicity and cellular uptake of PSA and HPSA nanoparticles were tested in A549 cells. In vivo cancer targeting of HPSA nanoparticles was evaluated by labeling PSA and HPSA nanoparticles with Cy5.5, a near-infrared fluorescent dye, for imaging. HPSA nanoparticles showed improved cancer-targeting ability compared with PSA. Doxorubicin-loaded HPSA (DOX-HPSA) nanoparticles were prepared using a simple dialysis method. An analysis of the in vitro drug-release profile and drug-delivery behavior showed that DOX was effectively released from DOX-HPSA nanoparticles. In vivo cancer therapy with DOX-HPSA nanoparticles in mice showed antitumor effects that resembled those of free DOX. Moreover, DOX-HPSA nanoparticles had low toxicity toward other organs, reflecting their tumor-targeting property. Hence, HPSA nanoparticles are considered a potential nanocarrier for anticancer agents.

Shikonin as an inhibitor of the LPS-induced epithelial-to-mesenchymal transition in human breast cancer cells.

Shikonin (SK), a natural naphthoquinone isolated from the Chinese medicinal herb, has been known to suppress the proliferation of several cancer cells. However, its role in the epithelial mesenchymal transition (EMT) has yet to be demonstrated. The aim of the present study was to examine the effects of SK on EMT. Lipopolysaccharide (LPS) induced EMT-like phenotypic changes, enhancing cell migration and invasion. SK markedly reduced the expression of the LPS-induced EMT markers, including N-cadherin in MDA-MB­231 cells, and increased the expression of E-cadherin in MCF-7 cells. SK also inhibited cell migration and invasion in vitro. The effects of SK on the LPS-induced EMT were mediated by the inactivation of the NF-κB-Snail signaling pathway. The results provided new evidence that SK suppresses breast cancer cell invasion and migration by inhibiting the EMT. Therefore, SK is a potentially effective anticancer agent for breast tumors, by inhibiting metastasis.

Suppression of LPS-induced epithelial-mesenchymal transition by aqueous extracts of Prunella vulgaris through inhibition of the NF-κB/Snail signaling pathway and regulation of EMT-related protein expression.

Epithelial-mesenchymal transition (EMT) is a pivotal event in the invasion and metastasis of cancer cells. Prunella vulgaris (PV) inhibits the proliferation of various cancer cells; however, its possible role in EMT has not been demonstrated. In the present study, we explored the effect of PV aqueous extract (PVAE), a typical medicine for decoction, on EMT. Lipopolysaccharide (LPS) induced EMT-like phenotype changes in cancer cell lines that enhanced cell migration and invasion. PVAE markedly inhibited these effects and produced accompanying changes in the expression of EMT markers, including decreased expression of N-cadherin and vimentin, and increased expression of ß-catenin. We found that PVAE effects on LPS-induced EMT were mediated by inhibition of the NF-κB/Snail signaling pathway. Our findings provide new evidence that PVAE suppresses cancer invasion and migration by inhibiting EMT. Therefore, we suggest that PVAE is an effective dietary chemopreventive agent with antimetastatic activity against malignant tumors.

Aqueous extract of Psoralea corylifolia L. inhibits lipopolysaccharide-induced endothelial-mesenchymal transition via downregulation of the NF-κB-SNAIL signaling pathway.

The epithelial-mesenchymal transition (EMT) is a pivotal event in the invasion and metastasis of cancer cells. Psoralea corylifolia L. (PC) inhibits the proliferation of various cancer cells. However, its possible role in EMT has not been identified. In the present study, we examined the effects of an aqueous extract of Psoralea corylifolia L. (PCAE), a typical medicinal decoction, on the EMT. Lipopolysaccharide (LPS) induced EMT-like phenotypic changes, enhancing cell migration and invasion. However, PCAE markedly reduced the expression of the LPS-induced EMT markers, including N-cadherin and vimentin, and increased the expression of ß-catenin. PCAE also inhibited cell migration and invasion in vitro. The effects of PCAE on the LPS-induced EMT were mediated by the inactivation of the NF-κB-SNAIL signaling pathway. The results provide new evidence that PCAE suppresses cancer cell invasion and migration by inhibiting EMT. Therefore, PCAE is a potentially effective dietary chemopreventive agent for malignant tumors since it inhibits metastasis.

Hispolon inhibits the growth of estrogen receptor positive human breast cancer cells through modulation of estrogen receptor alpha.

Human estrogen receptor α (ERα) is a nuclear transcription factor that is a major therapeutic target in breast cancer. The transcriptional activity of ERα is regulated by certain estrogen-receptor modulators. Hispolon, isolated from Phellinus linteus, a traditional medicinal mushroom called Sanghwang in Korea, has been used to treat various pathologies, such as inflammation, gastroenteric disorders, lymphatic diseases, and cancers. In this latter context, Hispolon has been reported to exhibit therapeutic efficacy against various cancer cells, including melanoma, leukemia, hepatocarcinoma, bladder cancer, and gastric cancer cells. However, ERα regulation by Hispolon has not been reported. In this study, we investigated the effects of Hispolon on the growth of breast cancer cells. We found that Hispolon decreased expression of ERα at both mRNA and the protein levels in MCF7 and T47D human breast cancer cells. Luciferase reporter assays showed that Hispolon decreased the transcriptional activity of ERα. Hispolon treatment also inhibited expression of the ERα target gene pS2. We propose that Hispolon, an anticancer drug extracted from natural sources, inhibits cell growth through modulation of ERα in estrogen-positive breast cancer cells and is a candidate for use in human breast cancer chemotherapy.

Shikonin blocks migration and invasion of human breast cancer cells through inhibition of matrix metalloproteinase-9 activation.

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, has been reported to promote tumor cell death. However, there are few reports concerning its effect on metastasis-related cell invasion and migration behavior. In the present study, we investigated the effect of shikonin on human breast cancer invasion and migration. We found that shikonin inhibited phorbol 12-myristate 13-acetate (PMA)-induced cell migration and invasion in MCF-7 breast cancer cells, which was correlated with modulation of matrix metalloproteinase-9 (MMP-9) through suppression of both expression and proteolytic and promoter activity. We also found that shikonin inhibited both MMP-9 expression and promoter activity in MDA-MB­231 cells with high metastatic potential. These results indicated that shikonin induces the suppression of migration and invasion through modulation of MMP-9 in human breast cancer cells. Therefore, shikonin may be a potential anticancer drug for human breast cancer therapy.

Shikonin inhibits the growth of human prostate cancer cells via modulation of the androgen receptor.

Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been shown to possess tumor cell killing activity. The human androgen receptor (AR) is a nuclear transcription factor that serves as a major therapeutic target for prostate cancer. However, AR regulation by shikonin has not been reported. We investigated the effects of shikonin on the growth of prostate cancer cells. We observed that shikonin decreased the expression of AR at both the mRNA and the protein levels in LNCaP and 22RV1 human prostate cancer cells. The results from a luciferase assay showed that shikonin decreased the transcriptional activity of AR. Moreover, shikonin treatment inhibited AR target gene expression, PSA and growth inhibition of prostate cancer cells. In conclusion, the present study shows for the first time that shikonin treatment causes transcriptional repression of AR and inhibition of its nuclear localization in human prostate cancer cells. We propose that shikonin, an anticancer drug extracted from natural sources, induces inhibition of cell growth through modulation of AR in androgen-responsive prostate cancer cells and is a candidate for use in cancer chemotherapy for human prostate cancer.