Aminolevulinic acid (ALA) is a prodrug that is converted in the heme biosynthesis pathway to protoporphyrin IX (PpIX) for photodynamic therapy (PDT) and fluorescence-guided tumor detection and resection. Although ALA-based therapy has been clinically used for the treatment of various types of tumors, clinical outcomes of ALA applications are not satisfactory due to issues such as low tumor PpIX production, high PpIX fluorescence heterogeneity, and low tumor to normal fluorescence contrast. We argue that a personalized ALA-based therapy would overcome these limitations and result in enhanced therapeutic outcomes. Since PpIX is endogenously produced in the complex heme biosynthesis pathway composed of 4 cytoplasmic and 4 mitochondrial enzymes and subject to cell membrane transporters, ALA-based therapy needs to be tailored to tumor phenotypic and genotypic characteristics that affect tumor PpIX production and accumulation. We found that genetic alterations in heme biosynthesis enzymes in tumor cells could cause significant changes in ALA-PpIX production. In these tumors, a low dose of ALA was able to achieve better PpIX fluorescence contrast between tumor and normal cells than a high dose of ALA that is commonly used. For tumors with elevated transporter activity, combination of ALA and a clinical transporter inhibitor is necessary for increasing ALA-PpIX fluorescence and reducing PpIX fluorescence heterogeneity. Overall we hope to demonstrate that a personalized ALA protocol optimized to fit tumor phenotype and genotype offers better treatment outcomes than applying ALA based on a one-size-fits-all approach.
Photodynamic therapy (PDT) involves the combination of a photosensitizer and light of a specific wavelength. Upon light activation in the presence of oxygen, photosensitizer molecules generate reactive oxygen species that cause cytotoxicity by inducing oxidative stress. Aminolevulinic acid (ALA) is a pro-drug used for the diagnosis and PDT treatment of various solid tumors based on endogenous production of heme precursor protoporphyrin IX (PpIX). Although nearly all types of human cells express heme biosynthesis enzymes and produce PpIX, tumor cells are found to have more PpIX production and accumulation than normal cells, allowing for the detection and treatment of solid tumors.
The objective of my research is to explore therapeutic approaches to enhance ALA-based tumor detection and therapy. We have found that high ABCG2 transporter activity in triple negative breast cancer cells (TNBC) contributed to reduced PpIX levels in cells, causing them to be more resistant towards ALA-PDT. The administration of an ABCG2 inhibitor, Ko143, was able to reverse cell resistance to ALA-PDT by enhancing PpIX mitochondrial accumulation and sensitizing cancer cells to ALA-PDT. Ko143 treatment had little effect on PpIX production and ALA-PDT in normal and ER- or HER2-positive cells. Furthermore, since some tyrosine kinase inhibitors (TKI) are known to block ABCG2 transporter activity, we screened a panel of tyrosine kinase inhibitors to examine its effect on enhancing PpIX fluorescence and ALA-PDT efficacy. Several TKIs including lapatinib and gefitinib showed effectiveness in increasing ALA-PpIX fluorescence in TNBC leading to increased cell death after PDT administration. These results indicate that inhibiting ABCG2 transporter using TKIs is a promising approach for targeting TNBC with ALA-based modality.
Vascular targeted photodynamic therapy is a promising cancer treatment modality by ablating tumor vasculature. The effectiveness of this treatment is often compromised by regrowth of endothelial cells, which causes tumor recurrence. In this preliminary report, we showed that activated PI3K signaling was involved in endothelial cell regrowth after PDT with verteporfin and combination between verteporfin-PDT and PI3K pathway inhibitor BEZ235 induced more cell apoptosis and greater inhibition in cell proliferation. These results suggest that rational combination of verteporfin-PDT and PI3K inhibitors result in enhanced treatment outcomes.
Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from
oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the
treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and
manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC
mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was
primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of
photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to
mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER
stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER
stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.
The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this
signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer.
We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell
line (PC3) and SV40-transformed mouse endothelial cell line
(SVEC-40). Combination of PDT and BEZ 235 (BEZ), a
pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of
sub-lethal PDT in both cell lines. The combination of
the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive
manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed
apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and
LY rapidly after treatment. Compared to SVEC, PC3 cells are
apoptosis-deficient and apoptosis was not significantly
enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be
enhanced by combination, depending on PI3K inhibitor and dose.
Vascular targeting photodynamic therapy (vPDT) is currently in clinical trial for prostate cancer (PCa)
treatment. In order to study the effect of vPDT on tumor metastasis, GFP-PC3 or PC-3 xenografts were
treated with verteporfin (BPD) PDT. Vascular function was assessed by ultrasound imaging; lymph node and
lung metastasis were assessed by fluorescence imaging. vPDT significantly reduced tumor blood flow within
30minutes to 2 hours of treatment. Sub-curative treatment resulted in re-perfusion within 2 weeks of treatment
and increased lymph node metastasis. With curative doses, no metastasis was observed.
In order to identify cellular or matrix factors and cytokines implicated, conditioned medium from BPD PDTtreated
endothelial cells was incubated with PC3 cells in vitro. Tumor cell proliferation and migration was
assessed. By immunoblotting, we evaluated the change in mediators of intracellular signaling or that may
determine changes in tumor phenotype. Low sub-curative dose (200ng/ml BPD) of endothelial cells was
associated with ~15% greater migration in PC3 cells when compared with control. This dose was also
associated with sustained activation of Akt at Ser 473, an upstream effector in the Akt/ mTOR pathway that
has been correlated with Gleason scores in PCa and with survival and metastasis in vitro and in vivo. In
conclusion, the study implicates efficacy of PDT of endothelial cells as an important determinant of its
consequences on adjacent tumor proliferation and metastasis.
Tumor vasculature is an attractive target for cancer therapy due to its accessibility to blood-borne therapeutic agents and
the dependence of tumor cells on a functional blood supply for survival and growth. Vascular targeting photodynamic
therapy (vPDT) is a novel modality based on the selective laser light activation of photosensitizers localized inside tumor
vasculature to shutdown tumor vascular function. Although this vascular targeting therapy is showing great promise for
cancer treatment, tumor recurrence has been observed in both preclinical and clinical studies. In this study, we intend to
enhance the therapeutic outcome of vascular targeting PDT by combining it with combretastatin A4 phosphate (CA4P), a
blood flow inhibitor. We found that the combination of CA4P and vPDT significantly increased endothelial cell
apoptosis than each single therapy. Western blot analysis suggests that myosin light chain kinase (MLCK) is a common
target of CA4P and vPDT. In a PC-3 prostate tumor model, we found that CA4P was able to greatly enhance tumor
response to vPDT. These results demonstrate that CA4P and vPDT can be combined to enhance the therapeutic effect.