Original ArticleTumor vessel normalization by the PI3K inhibitor HS-173 enhances drug delivery
Introduction
Tumor angiogenesis, the formation of new blood vessel, plays an essential role in tumor progression [1], [2]. Extensive angiogenesis in tumors is accompanied by a defective vascular architecture, leading to increased vascular permeability, hypoxia, and high interstitial fluid pressure. Cancer growth strictly depends on the expansion of the host vasculature, which not only supplies oxygen and nutrients to the tumor tissue, but also provides cancer cells with a metastatic route for colonizing distant organs [3], [4], [5]. Therefore, angiogenesis represents a critical process during tumor initiation and malignant progression, and is a target of anticancer drugs. Some studies have shown that inhibition of angiogenesis suppresses metastasis and tumor growth [6], [7], whereas others have reported that it is associated with enhanced intratumoral hypoxia, increased local tumor invasion, and frequency of metastasis [7], [8]. Indeed, the benefits of antiangiogenic agents are transient, and the initial response is often followed by the establishment of resistance and escape mechanisms, leading to tumor relapse [7], [9]. These observations accentuate the need for a more comprehensive understanding of the biological processes that underlie tumor vascularization and set the stage for additional angiogenesis-targeted therapies.
Compared with normal vessels, tumor vessels are heterogeneous and tortuous, branch chaotically, and have an uneven vessel lumen. They also frequently lack pericyte coverage and show an abnormal basement membrane, resulting in vascular instability and altered permeability. These vessel abnormalities generate a promalignant microenvironment that can facilitate the dissemination of cancer cells through leaky vessels, thus causing poor response to therapy [10]. In addition, normal vessels are composed of a monolayer of interconnected adherent endothelial cells (ECs), whereas tumor vessel ECs can detach from the basement membrane and vessels, resulting in pericyte loss [11]. Namely, the structural stability of vessels is dependent on both the distribution of ECs and pericyte coverage [12], [13], factors that are important in promoting vessel normalization.
These observations raise the question of whether tumor vessel normalization offers an alternative therapeutic strategy for reducing metastatic spread and enhancing tumor responses to chemotherapy and radiotherapy [14]. Recent studies have typically used ‘vessel blocking’ to describe a situation in which the total number of immature and mature vessels is reduced, and ‘vessel normalization’ in case where only immature vessels are removed and the remaining vessels become more normal and mature [15]. Thus, in the process of vessel normalization, the architecture of the remaining vasculature is largely restored, thereby reducing vessel permeability and improving tumor blood flow [15], [16], [17].
The effects of vessel normalization may be even more important for intratumoral delivery of drugs [18]. Several preclinical and clinical studies have indicated that antiangiogenic drugs lead to vessel normalization in addition to causing anti-vascular effects [19], [20]. One example is bevacizumab, which is mainly given in combination with chemotherapy. The beneficial effect of this combination therapy is attributable to vessel normalization and the consequent increase in tumor uptake of co-delivered chemotherapeutic agents [21]. However, most anti-angiogenic drugs, such as vascular endothelial growth factor receptor (VEGFR) inhibitors, have failed to optimize drug-dose and interval for the combination, such that the drug induces metastasis through increased vessel leakiness and destruction of ECs [22].
A number of previous studies have reported that phosphoinositide 3-kinase (PI3K) signaling affects tumor angiogenesis both directly and indirectly [23]. PI3K activation also upregulates VEGF and promotes vessel formation [24]. Thus, inhibition of the PI3K pathway suppresses tumor cells and affects the tumor stroma, including the vasculature. Indeed, targeting PI3K signaling has been shown to induce antiangiogenic activity in preclinical tumor models [25], [26], [27], [28]. In addition, PI3K inhibitors block neovascularization and downregulate VEGF, and also inhibit tumor growth [29], [30], [31]. Recently, the PI3K inhibitor BEZ235 has been shown to increase blood perfusion in normalized tumor vessels, resulting in an antiproliferative effect by counterbalancing the increased tumor growth [32]. Similarly, the EGFR inhibitor erlotinib has shown improved delivery of cisplatin and synergistic inhibition of tumor growth by increasing perfusion. These previous studies suggest that these drugs produce a more durable induction of vascular normalization. However, only a limited number of studies have investigated the effects of PI3K inhibitors on vessel normalization and its mechanisms are not well understood.
In this study, we investigated the potential of HS-173, a novel PI3K inhibitor, as well as BEZ235 to inhibit tumor growth by inducing vascular normalization. We found that HS-173 and BEZ235 caused vessel normalization in tumors, inhibiting vascular leakage, improving capillary integrity, and increasing vessel density. This was particularly notable for HS-173 in combination therapy, which increased blood perfusion and led to delayed tumor growth. Therefore, vessel normalization by HS-173 could be a novel therapeutic strategy for inhibiting tumor growth by improving drug delivery to the tumor.
Section snippets
Cell culture
MIA PaCa-2 and B16 cells were purchased from ATCC (Manassas, VA), and human umbilical vein endothelial cells (HUVECs) were obtained from Merck Millipore (Temecula, CA). MIA PaCa-2 and B16 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). DMEM, FBS and P/S were purchased from Invitrogen (Life Technologies, Grand Island, NY). HUVECs were cultured in 2% gelatin-coated dishes in M199 medium
Materials
Ethyl 6-(5-(phenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate (HS-173) was synthesized as previously described [33]. NVP-BEZ235 (BEZ235) was purchased from LC laboratories (Woburn, MA). Doxorubicin was purchased from Sigma-Aldrich (Munich, Germany). HS-173 and BEZ235 were dissolved in dimethyl sulfoxide (DMSO), and doxorubicin (DOX) was dissolved in distilled water.
PI3K inhibitors decrease tumor growth and metastasis in animal models
To evaluate the effects of PI3K inhibitors on tumor growth, MIA PaCa-2 cells or B16 cells were inoculated to Balb/c nude mice. When tumor sizes reached approximately 50–100 mm3, mice were injected with 30 mg/kg of HS-173 or BEZ235, or vehicle (control) (Fig. 1A). Tumor growth was reduced by 50% and 29% by HS-173 and BEZ235, respectively, compared with controls in the MIA PaCa-2 model, and by 52% and 25% in the B16 model (Fig. 1B). Similar effects were obtained by inhibiting expression of p-Akt
Discussion
HS-173 has shown anticancer effects through inhibition of PI3K/Akt signaling in tumors. We have also previously shown that HS-173 exerts its antiangiogenic efficacy by blocking tumor vessels [33], [36], [37]. However, it is not yet known whether the anticancer effects of HS-173 are solely attributable to blockage of tumor vessels. Recent studies on angiogenesis therapy have emphasized the importance of vessel normalization for drug delivery and long-term efficacy. Thus, the
Acknowledgements
This research was supported by the National Research Foundation (NRF) Grant (2015M3A9C4075818, 2015R1A2A1A10054108, 2014009392, 2014M3C1A3051476) and the Korea Health Technology R&D Project, Ministry of Health and Welfare (HI15C0554), Republic of Korea.
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These authors equally contributed to this work.