br models unsurprisingly showed variable sensitivities to mi
models unsurprisingly showed variable sensitivities to miR-1291 and Gem-nP treatment alone. The third PDX, PA-0327, seemed to be the most invasive, showed lower sensitivity to both Gem-nP and miR-1291 monotherapy. Generally, combination treatment with miR-1291 and Gem-nP reduced the final tumor sizes of PA-0327 by > 80%, supporting combination treatment including dose tailoring as an optimal strategy to combat PC. Nevertheless, subcutaneous PDX mouse models used in current study might not be as comprehensive and persuasive as ortho-topic tumor models when considering pharmacokinetics properties of a drug, although subcutaneous PDX animal models are able to re-capitulate the histologic and genotypic characteristics of human tumor and indicative of tumor responses to therapeutic drugs. To better de-monstrate the pharmacokinetics and pharmacodynamics as well as therapeutic potential of miR-1291 mono- and combination treatment, further studies are highly warranted by using orthotopic PDX animal models before clinical investigation.
Optimal outcomes of miR-1291 plus Gem-nP combination treatment are inevitably due to multi-targeting in PC cells. Consistent with our previous findings , current study showed that miR-1291 alone en-hanced apoptosis, as manifested by higher c-caspase-3/7 levels, which increased to even greater degrees when Gem-nP was co-administered. Gemcitabine is a nucleoside analogue that is converted to gemcitabine triphosphate, and subsequently inhibits DNA synthesis by incorporating into DNA, leading to G1/S aminonucleoside arrest and apoptosis [36,37].
Fig. 5. Bioengineered miR-1291 prodrug monotherapy and combination therapy with Gem-nP in PDX mouse model derived from clinical PC tissues (PA-0387). (A) PDX tumor growth was significantly suppressed by miR-1291 monotherapy or combination therapy, as compared to MSA or buﬀer control. *P < 0.05, **P < 0.01, and ***P < 0.001 (2-way ANOVA with Bonferroni post-tests). (B) Comparison of dissected tumor from mice with diﬀerent treatments. (C) Weights of the dissected xenograft tumors. **P < 0.01, ***P < 0.001 (1-way ANOVA). (D) Representative IHC of PDX tumors stained with Ki-67 or cleaved caspase-3 antibodies. Combination treatment induced the highest degree of apoptosis (Red arrow: caspase-3 staining) while cell proliferation did not diﬀer much among diﬀerent treatment groups. Scale bar indicates 100 μm. (E) Body weights were not diﬀerent from each treatment. (F) Blood biochemistry profiles were not altered by any drug treatment. Values are mean ± SD (N = 5 per group, except N = 3 for blood chemistry profiles). The ranges of individual markers were marked as references. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Paclitaxel reduces cell mitosis through stabilization of microtubes . As indicated by the increase in γH2A.X and H3PS10 levels, actions of Gem-nP on DNA damage and mitotic arrest were obvious in PC cell lines. Likewise, co-administration of miR-1291 enhanced the levels of DNA damage and mitotic arrest, providing a good explanation of the sensitization of PC cells to Gem-nP by miR-1291. It is also notable that AsPC-1 cells were more sensitive to miR-1291 and chemotherapies than PANC-1 cells, in agreement with the finding on more striking increases in the expression of marker proteins at earlier time points (48 h post-treatment) in AsPC-1 cells.
The present study also validated a new target for miR-1291, ARID3B, an addition to those reported previously [17,19–22]. While a miRNA generally reduces target gene expression, ARID3B was rather upregulated in PC cells by miR-1291. Although the precise mechanisms are unknown, there is growing evidence that miRNAs are also able to stimulate the expression of target genes through direct or indirect ac-tions . In addition, the role of ARID3B in cancer remains con-troversial although there are only a limited number of reports. Some studies showed that ARID3B promoted cancer cell proliferation or tu-morigenesis [40–43], whereas other studies demonstrated that ARID3B played an important role in the induction of apoptosis [24,44]. These
studies diﬀer much in the types of cancer cells investigated and reagents used, as well as study designs. The upregulation of ARID3B by miR-1291 not only agrees with the function of ARID3B in the induction of apoptosis showed by others [24,44] but also the role of miR-1291 in the enhancement of apoptosis in PC cells reported by us very recently . Therefore, the upregulation of pro-apoptotic ARID3B is likely one of many possible mechanisms behind the antitumor function of miR-1291, and further studies are warranted to improve our understanding of ARID3B functions in PC.