Full length articleJanus nanocarrier-based co-delivery of doxorubicin and berberine weakens chemotherapy-exacerbated hepatocellular carcinoma recurrence
Graphical abstract
Introduction
Hepatocellular carcinoma (HCC) leads to nearly millions of deaths worldwide each year with an increasing mortality rate [1,2]. Advances in chemotherapy partially alleviates the clinical outcomes of HCC patients [3], [4], [5]. Unfortunately, more than ninety percent of patients suffer from recurrence after treatment, which majorly accounts for the morbidity [6], [7], [8], [9]. This fundamental challenge urges clinicians to re-evaluate the application of current cell death-based strategies and to investigate the molecular mechanisms in depth [10,11]. To enhance chemotherapeutic agent-triggered cancer cell apoptosis and to caspase elicits repair, our group and others have recently reported that caspase-3-dependent cleavage of cytosolic calcium-independent phospholipase A2 (iPLA2) during chemotherapy triggers the release of arachidonic acid (AA) and prostaglandin E2 (PGE2). The activation of the AA metabolic pathway leads to the repopulation of surviving cancer cells, ultimately promoting recurrence [12]. Therefore, we speculated that the balance between apoptotic cell death and caspase-driven tumor cell survival and repopulation could be used for effective tumor killing and death-related recurrence inhibition [12], [13], [14]. Among several important targets involved in the ``Phoenix Rising'' signaling pathway [15], [16], [17], [18], iPLA2 acts as a key link between caspase activation and the AA metabolic pathway. We previously demonstrated that berberine (BER), which is a competitive inhibitor of PLA2 [19], [20], [21], inhibits chemotherapy-induced repopulation of ovarian cancer cells by suppressing the AA metabolic pathway and FAK phosphorylation [22]. However, the role of the ``Phoenix Rising'' signaling pathway in chemotherapy-exacerbated HCC recurrence and the effects of BER on this event remain elusive. Therefore, we hypothesized that the combination of a chemotherapeutic agent and the ``Phoenix Rising'' signaling pathway inhibitor might inhibit cell death-induced HCC recurrence, thereby promoting effective chemotherapeutic outcomes.
To achieve synergic effect on chemotherapy-exacerbated HCC recurrence with minimal side effects, drug codelivery system is highly desirable for imaging-guided HCC-targeted and controlled delivery. Chemotherapeutic agent doxorubicin (DOX) and the ``Phoenix Rising'' signaling pathway inhibitor BER were exploited in this co-delivery system. Magnetic mesoporous silica nanoparticles (M-MSNs) have attracted considerable attention as promising carriers due to their good biocompatibility, high drug loading, surface modification versatility, and magnetic properties for magnetic resonance imaging (MRI) contrast [23], [24], [25], [26]. Our group has recently used Janus-type magnetic mesoporous silica nanoparticles (Janus M-MSNs) with an architectural advantage of minimal interfering [23]. Continuous efforts have demonstrated that our drug delivery systems exhibited stronger magnetic properties, a higher intracellular internalization and more tumor accumulation in comparison to traditional core-shell structures such as M-MSNs [27], which could further benefit magnetically targeted chemotherapy and MRI-guided, hyperthermia-enhanced gene therapy of HCC [28], [29], [30]. Furthermore, due to the high expression level of the CD44 receptor on HCC cells surface [31,32], hyaluronic acid (HA), a biodegradable, biocompatible, and nonimmunogenic linear polysaccharide, was employed as a bifunctional ligand to prolong blood circulation and improve the accumulation of nanoparticles in HCC foci [33], [34], [35]. In such a scenario, HA acts as a targeting component due to its high affinity for CD44 receptor, which facilitating the CD44 receptor-mediated endocytosis of HA-modified nanocarriers in HCC cells. Therefore, HA-conjugated Janus M-MSNs with MR imaging and HCC-targeting behavior can be exploited as an ideal co-delivery system for efficient and safe HCC therapy and reducing chemotherapy-exacerbated recurrence [36], [37], [38].
In this study, we first validate our hypothesis of chemotherapy promotes HCC recurrence in clinical samples and a murine repopulation model. Then, we aimed to explore the crucial role of the activated caspase-3-iPLA2-COX-2 pathway in the HCC cell repopulation model. We illustrated BER significantly reverses doxorubicin (DOX)-induced HCC repopulation both in vitro and in vivo. To improve the drug delivery efficiency of DOX and BER and to reduce the side effects of DOX, the co-delivery system of HA-conjugated Janus M-MSNs (HA-MSN@DB) was developed. HCC cell-targeting, pH-sensitive drug release and anticancer properties of HA-MSN@DB were assessed in CD44-overexpressing HCC cells and in normal liver cells. MR imaging, biodistribution, biocompatibility, tumor inhibition and recurrence studies were subsequently performed in H22 tumor-bearing mice. The ``Phoenix Rising'' signaling pathway during DOX-induced HCC repopulation was analyzed in vivo. Our findings prove the co-delivery of DOX and BER via HA-conjugated Janus M-MSNs, which is a promising, efficient and safe HCC chemotherapeutic strategy with decreased recurrence.
Section snippets
Cell culture and co-culture system
Cancer cell lines, including HepG2 human hepatocellular carcinoma cells and H22 mouse hepatoma cells, and normal cell lines, including HL-7702 human embryonic liver cells and NIH-3T3 mouse fibroblast cells, were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 U/mL streptomycin at 37 °C in a 5% carbon dioxide atmosphere. The cells were passaged using 0.25% trypsin/EDTA. The cell lines that were used in this study were obtained from ATCC.
Coculture
Chemotherapy is a risk factor for clinical HCC recurrence
To investigate the relationship between chemotherapy and HCC recurrence in the clinic, we collected and analyzed data from a total of 67 patients with HCC. At baseline, the mean (±SD) age of patients with HCC with recurrence was 57.85 ± 9.56 years. There were significant differences in portal vein thrombosis, BCLC stage and chemotherapy between the two groups of patients. There were no significant differences in the other variables between the two groups. Table S1 shows a summary of the study
Discussion
An in-depth understanding of the HCC recurrence mechanism is essential for developing efficient treatment strategies [40,41]. We conducted a clinical study in patients with HCC with and without recurrence and identified that chemotherapy is a risk factor for HCC recurrence. Although cytotoxic chemotherapeutics kill cancer cells, cancer recurrence nearly inevitably occurs [40]. Increasing evidence has suggested that cancer recurrence after chemotherapy is partially attributed to
Conclusion
In summary, we revealed the crucial role of the activated caspase-3-iPLA2-COX-2 pathway during DOX-exacerbated HCC recurrence and demonstrated that BER significantly limits DOX-triggered growth-stimulating properties in vitro and in vivo. We developed HA-modified MSNs as multifunctional drug carriers and coloaded DOX and BER in the mesoporous silica framework. The MSNs and HA in HA-MSN@DB led to pH-responsive drug release and CD44 receptor-mediated HCC targeting, respectively. Therefore,
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Grand Nos. 81371681, 81771982, 81601609, 61535010, and 8160071152), the National Key Research and Development Program of China (Grand Nos. 2017YFF0108600 and 2016YFF0103800), the Natural Science Foundation of Jiangsu Province (No. BE2015601) and the Science and Technology Department of Suzhou City (Nos. SS201539 and ZXY201434).
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These authors contributed equally to this work.