Targeting cancer cell integrins using gold nanorods in photothermal therapy inhibits migration through affecting cytoskeletal proteins.

Metastasis is responsible for most cancer-related deaths, but the current clinical treatments are not effective. Recently, gold nanoparticles (AuNPs) were discovered to inhibit cancer cell migration and prevent metastasis. Rationally designed AuNPs could greatly benefit their antimigration property, but the molecular mechanisms need to be explored. Cytoskeletons are cell structural proteins that closely relate to migration, and surface receptor integrins play critical roles in controlling the organization of cytoskeletons. Herein, we developed a strategy to inhibit cancer cell migration by targeting integrins, using Arg-Gly-Asp (RGD) peptide-functionalized gold nanorods. To enhance the effect, AuNRs were further activated with 808-nm near-infrared (NIR) light to generate heat for photothermal therapy (PPTT), where the temperature was adjusted not to affect the cell viability/proliferation. Our results demonstrate changes in cell morphology, observed as cytoskeleton protrusions-i.e., lamellipodia and filopodia-were reduced after treatment. The Western blot analysis indicates the downstream effectors of integrin were attracted toward the antimigration direction. Proteomics results indicated broad perturbations in four signaling pathways, Rho GTPases, actin, microtubule, and kinases-related pathways, which are the downstream regulators of integrins. Due to the dominant role of integrins in controlling cytoskeleton, focal adhesion, actomyosin contraction, and actin and microtubule assembly have been disrupted by targeting integrins. PPTT further enhanced the remodeling of cytoskeletal proteins and decreased migration. In summary, the ability of targeting AuNRs to cancer cell integrins and the introduction of PPTT stimulated broad regulation on the cytoskeleton, which provides the evidence for a potential medical application for controlling cancer metastasis.

Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm2) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome c and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.

Simultaneous Time-Dependent Surface-Enhanced Raman Spectroscopy, Metabolomics, and Proteomics Reveal Cancer Cell Death Mechanisms Associated with Gold Nanorod Photothermal Therapy.

In cancer plasmonic photothermal therapy (PPTT), plasmonic nanoparticles are used to convert light into localized heat, leading to cancer cell death. Among plasmonic nanoparticles, gold nanorods (AuNRs) with specific dimensions enabling them to absorb near-infrared laser light have been widely used. The detailed mechanism of PPTT therapy, however, still remains poorly understood. Typically, surface-enhanced Raman spectroscopy (SERS) has been used to detect time-dependent changes in the intensity of the vibration frequencies of molecules that appear or disappear during different cellular processes. A complete proven assignment of the molecular identity of these vibrations and their biological importance has not yet been accomplished. Mass spectrometry (MS) is a powerful technique that is able to accurately identify molecules in chemical mixtures by observing their m/z values and fragmentation patterns. Here, we complemented the study of changes in SERS spectra with MS-based metabolomics and proteomics to identify the chemical species responsible for the observed changes in SERS band intensities during PPTT. We observed an increase in intensity of the bands at around 1000, 1207, and 1580 cm-1, which were assigned in the literature to phenylalanine, albeit with dispute. Our metabolomics results showed increased levels of phenylalanine, its derivatives, and phenylalanine-containing peptides, providing evidence for more confidence in the SERS peak assignments. To better understand the mechanism of phenylalanine increase upon PPTT, we combined metabolomics and proteomics results through network analysis, which proved that phenylalanine metabolism was perturbed. Furthermore, several apoptosis pathways were activated via key proteins (e.g., HADHA and ACAT1), consistent with the proposed role of altered phenylalanine metabolism in inducing apoptosis. Our study shows that the integration of the SERS with MS-based metabolomics and proteomics can assist the assignment of signals in SERS spectra and further characterize the related molecular mechanisms of the cellular processes involved in PPTT.