RING finger protein 43 (
RNF43
) is a ubiquitin E3 ligase that negatively regulates Wnt/β-catenin signalling. Mutation, inactivation and downregulation of
RNF43
in cholangiocarcinoma (CCA) are associated with a less favourable prognosis. Since the functional role of RNF43 in CCA has not yet been demonstrated, the present study aimed to assess the effect of its overexpression in mediating CCA suppression via Wnt/β-catenin signalling pathway inhibition. Accordingly,
RNF43
was overexpressed, and various malignant phenotypic changes studied, including cell proliferation, cell migration, chemotherapeutic sensitivity and the expression of several Wnt/β-catenin target genes. Overexpression of RNF43 in the CCA cell-line KKU-213B hindered activation of Wnt/β-catenin signalling, evidenced by: i) Accumulation of β-catenin in the cytoplasmic fraction and downregulation of several known Wnt target genes at the mRNA level [
AXIN2
, survivin (
BIRC5
),
CCND1, MMP-7, c-MYC
and
ABCB1
(
MDR1
)]; ii) a reduction of cell proliferation; iii) a significant decrease in KKU-213B cell migration with RNF43 overexpression via upregulation of E-cadherin (
CDH1
); and iv) a reduction in N-cadherin (
CDH2
),
MMP-2, MMP-7
and
MMP-9
. In addition, overexpression of RNF43 increased 5-fluorouracil sensitivity and downregulation of ABC transporter genes [including
ABCB1
and
ABCC1
(MRP1)]. The current results demonstrate a functional role for RNF43 in CCA by: i) Blocking β-catenin nuclear translocation; and ii) the subsequent downregulation of Wnt/β-catenin target genes (the latter being involved in the progression of CCA and chemotherapeutic drug susceptibility). Therefore, the present findings suggest that RNF43 could serve a tumour suppressive role in CCA.
To resurrect antibacterial efficacy of colistin (CLT), ceftazidime (CAZ) and cefotaxime (CTX), Stephania suberosa extract (SSE) was combined with these particular antibiotics to combat CLT‐resistant Enterobacter cloacae (CREC) isolates. Disc diffusion assay showed that SSE inhibited E. cloacae strains with the dose‐dependent manner. Minimum inhibitory concentrations (MICs) of SSE against all tested strains were 2000 µg ml−1. CREC DMST 37480 and 19719 were found to be resistant to CLT with MICs of 64 and 4 µg ml−1, respectively, and also resistant to CAZ. These strains showed a minimum bactericidal concentration (MBC) of SSE at 8000 µg ml−1. Checkerboard assay showed that CLT resistance was synergistically reversed by SSE against CREC DMST 37480 and 19719 with a fractional inhibitory concentration (FIC) indices of 0·253 and 0·265, respectively. Time‐killing assay confirmed synergistic interaction by a decline in the viability combined treated group compared to an individual. CREC DMST 19719 was found to produce AmpC β‐lactamase. SSE cannot resurrect CAZ in an AmpC producer. The scanning electron microscopy showed that SSE and CLT induced cell damages at different sites. GC‐MS analysis identified 25 known Phyto‐compounds. SSE and CLT combination could be further developed as a novel agent for treating multidrug‐resistant CREC.
Significance and Impact of the Study
Resistance to colistin (CLT), an alternative agent for treating multiple drug‐resistant Enterobacter cloacae, is among the most serious, life‐threatening issues. This study utilizes Stephania suberosa extract (SSE) to revive the antibacterial activity of colistin that has lost its antibacterial effectiveness in inhibiting E. cloacae. The findings support the development of the combined agent between SSE and colistin to conquer colistin‐resistant E. cloacae.
Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and the genes for glucocerebrosidases 1, 2, and 3 (GBA1, GBA2, and GBA3) were therefore studied in CCA. The biological functions of GCS for cell proliferation and cisplatin sensitivity in CCA were explored. GCS expression was higher in CCA tumor tissues than that of GBA1, GBA2, and GBA3. Verification of GCS expression in 29 paired frozen CCA tissues showed that 8 of 29 cases (27.6%) had high GCS expression. The expression of GCS and GBA2 was induced in CCA cell lines following low-dose cisplatin treatment. Suppression of GCS by either palmitoylamino-3-morpholino-1-propanol (PPMP), GCS knockdown or a combination of the two resulted in reduced cell proliferation. These treatments enhanced the effect of cisplatin-induced CCA cell death, increased the expression of apoptotic proteins and reduced phosphorylation of ERK upon cisplatin treatment. Taken together, inhibition of the GCS increased cisplatin-induced CCA apoptosis via the inhibition of the ERK signaling pathway. Thus, targeting GCS might be a strategy for CCA treatment.
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